Methods for analysis of gene expression

ABSTRACT

This invention provides methods, compositions and kits for gene expression analysis and gene expression profiling. The methods of the invention are highly sensitive; have a wide dynamic range; are rapid and inexpensive; have a high throughput; and allow the simultaneous differential analysis of a defined set of genes. The methods, compositions and kits of the invention also provide tools for gene expression data collection and relational data analysis.

CROSS-REFERENCES TO RELATED APPLICATIONS

[0001] This application claims priority to and benefit of U.S.application No. 60/179,006, filed Jan. 28, 2000, the full disclosure ofwhich is incorporated herein by reference.

STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSOREDRESEARCH AND DEVELOPMENT

[0002] The United States government may own rights in the presentinvention pursuant to grant numbers HG01700-02, R43-CA83382 andN43-ES-81006 from the National Institutes of Health.

BACKGROUND OF THE INVENTION

[0003] Functional genomics is a rapidly growing area of investigation,which includes research into genetic regulation and expression, analysisof mutations that cause changes in gene function, and development ofexperimental and computational methods for nucleic acid and proteinanalyses. The Human Genome Project has been the major catalyst drivingthis research; it has been through the development of high-throughputtechnologies that it has been possible to map and sequence complexgenomes. However, while the nucleic acid sequence information elicitedby these technologies represents the “structural” aspects of the genome,it is the interworkings of the genes encoded therein, and the geneproducts derived from these sequences, that will give a meaningfulcontext to this information. In particular, gene expression monitoringcan be utilized to examine groups of related genes, interlockingbiochemical pathways, and biological networks as a whole.

[0004] This rapidly growing set of cloned human genes provides aplethora of candidate drug targets for testing against complex chemicallibraries. In order to efficiently test the impact(s) of a large numberof putative drug compounds on the expression profile of one or more setsof genes, methods are needed that are sensitive, quantitative, extremelyrapid, and adaptable to automation, in order to be cost-effective.Present day technologies do not meet these demands. The presentinvention addresses this need by providing novel methods for analyzinggene expression, systems for implementing these techniques, compositionsfor preparing a plurality of amplification products from a plurality ofmRNA target sequences, and related pools of amplification products.

SUMMARY OF THE INVENTION

[0005] The present invention provides methods for analyzing geneexpression. The methods include obtaining a plurality of cDNA targetsequences, and multiplex amplifying these sequences, a process whichinvolves combining the plurality of target sequences with a plurality oftarget-specific primers and one or more universal primers, to produce aplurality of amplification products. The target sequences are obtainedin any of a number of manners, such as by performing reversetranscription on a set of mRNA molecules. The mRNA molecules areoptionally derived from cells, organisms, or cell cultures, which areoptionally exposed to one or more specific treatments that potentiallyalter the biological state of the cell, organism, or cell culture.

[0006] Target-specific primers for use in the methods of the presentinvention include oligonucleotides comprising a first sequence that isderived from a target gene of interest and positioned within a 3′ regionof the oligonucleotide, and a second sequence that is complementary to auniversal primer and positioned within the 5′ region of theoligonucleotide. The target specific primers can be categorized asforward primers or reverse primers, depending upon the relativeorientation whether the primer versus the polarity of the nucleic acidsequence (e.g., whether the primer binds to the coding strand or acomplementary (noncoding) strand of the target sequence).

[0007] The universal primers used in the methods of the presentinvention are sequences common to a plurality of target-specificprimers, but preferably not present in the template nucleic acid (i.e.,the plurality of target sequences). As such, a universal primertypically does not hybridize to the target sequence template during aPCR reaction. However, since the universal primer sequence iscomplementary to a portion of one or more target-specific primers usedin the present invention, the universal primer can initiatepolymerization using a target-specific primer-amplified product as atemplate. In some embodiments of the present invention, multipleuniversal primers having sequences distinct from one another areutilized; these universal primers are then called “semi-universal”primers. As one example, a plurality of semi-universal primers caninclude primer sequences that are complementary to one or more forwardtarget-specific primers, one or more reverse target-specific primers, ora combination thereof.

[0008] Optionally, the multiplex amplification process involvessimultaneously amplifying a plurality of cDNA molecules in the samereaction mixture. This can be achieved, for example, by employing one ormore target-specific primer pairs (where each pair comprising a forwardtarget-specific primer and a reverse target-specific primer) and one ormore universal primer pairs, (also comprising pairs of forward andreverse universal primers). In some embodiments of the presentinvention, the multiplex amplification involves providing the universalprimer in an excess concentration relative to the target-specificprimer.

[0009] In some embodiments of the methods of the present invention, thelength of one or more of the universal primers or target-specificprimers is altered prior to combination in the multiplex amplificationstep. This alteration in length can be achieved, e.g., by addingnucleotides to the end of the primer sequence, inserting nucleotideswithin the primer sequence, incorporating a non-nucleotide linker withinthe primer sequence, or cleaving a cleavable linkage within the primersequence. As one example, alteration of the length of a target-specificprimer is achieved by inserting nucleotides between the universalsequence portion (i.e., that sequence complementary to the universalprimer sequence) and the target-specific sequence of the primer.

[0010] One or more of the nucleic acid sequences used as universalprimers and target-specific primers in the methods of the presentinvention can optionally include a cleavable linkage or a non-nucleotidelinker as a sequence element. This non-nucleotide linker can include,e.g., non-cleavable linkages, alkyl chains, or abasic nucleotides.Furthermore, the nucleic acid sequences used as universal primers andtarget-specific primers in the methods of the present invention canoptionally include one or more labels. Labels for use in the methods ofthe present invention can include, e.g., a chromaphore, a fluorophore, adye, a releasable label, a mass label, an affinity label, a frictionmoiety, a hydrophobic group, an isotopic label, or a combinationthereof. The same label can be incorporated into disparate primers usedin a multiplexed amplification; alternatively, unique labels orcombination of labels can be associated with each member of theplurality of primers.

[0011] Furthermore, the multiplex amplification optionally includes areference sequence that contains a region homologous to at least onemember of the plurality of target-specific primers. The referencesequence (or sequences) can be endogenously present in the cDNAcontaining the target sequence, or it can be exogenously added to thecDNA sample.

[0012] One or more members of the plurality of amplification productsare separated by any of a variety of techniques known to those of skillin the art. In a preferred embodiment of the present invention, themembers are separated using one or more separation techniques, such asmass spectrometry, electrophoresis (using, for example, capillaryelectrophoresis, microcapillary electrophoresis, agarose and/oracrylamide gel platforms), chromatography (e.g., such as HPLC or FPLC),or various microfluidic techniques.

[0013] The one or more members are detected by any of a number oftechniques, thereby generating one or more sets of gene expression data.For example, in a preferred embodiment, the amplification products areseparated and detected by performing HPLC followed by mass spectroscopy.

[0014] Detection is performed, for example, by measuring the presence,absence, or quantity/amplitude of one or more properties of theamplification products. Example properties of the amplification productsinclude, but are not limited to, mass, light absorption or emission, andone or more electrochemical properties. In embodiments in which one ormore of the primers includes a label, the inherent property can bedependent upon the identity of the label. In one embodiment, detectionof the amplification products involves resolving a first signal from asingly labeled amplification product and a second signal from a singlelabeled (or multiply labeled) amplification product by deconvolution ofthe data. In an alternative embodiment, detection of the amplificationproducts involves resolving a first signal from a singly labeledamplification product and a second signal from a single or multiplylabeled amplification product by reciprocal subtraction of the first orsecond signal from an overlapping signal. Thus, one or moreamplification products are detected and the information collected isused to generate a set of gene expression data.

[0015] The set of gene expression data are stored in a database; thisdata is then used, e.g., to perform a comparative analysis (for example,by measuring a ratio of each target gene to each reference gene or otheranalysis of interest).

[0016] The present invention also provides methods for analyzing geneexpression including the steps of obtaining cDNA from a plurality ofsamples for a plurality of target sequences; performing a plurality ofmultiplexed amplifications of the target sequences, thereby producing aplurality of multiplexed amplification products; pooling the pluralityof multiplexed amplification products; separating the plurality ofmultiplexed amplification products; detecting the plurality ofmultiplexed amplification products, thereby generating a set of geneexpression data; storing the set of gene expression data in a database;and performing a comparative analysis of the set of gene expressiondata. As in the previous embodiments, a plurality of target-specificprimers and universal primers are employed in the multiplexedamplification step. Either the universal primer(s) or thetarget-specific primer(s) can be labeled. In one embodiment of thesemethods, a first multiplexed amplification is performed using a primerhaving a first label that produces a first signal, and a secondmultiplexed amplification is performed with a primer comprising a secondlabel that produces a second signal, wherein the first and secondsignals are distinguishable from one another.

[0017] In another embodiment, the plurality of amplification productsare detected by shifting the mobility of member amplification productsrelative to one another For example, amplification of the targetsequences is performed using universal primers having two or morelengths; detection of the plurality of multiplexed amplificationproducts produced using these primers involves measuring one or moresize shifts among the plurality of multiplexed amplification products.Alternatively, the method is performed using target-specific primershaving two or more lengths, leading to generation ofdifferentially-sized amplification products. The shift in size can beachieved, for example, by using primers having cleavable linkagesincorporated into their sequences. Alternatively, the shift in size canbe achieved by incorporation of a friction moiety into one or more ofthe universal primers, thereby creating a reduction in mobility of theamplification products.

[0018] The multiplex amplification reaction used in the methods of thepresent invention includes, but is not limited to, a polymerase chainreaction, a transcription-based amplification, a self-sustained sequencereplication, a nucleic acid sequence based amplification, a ligase chainreaction, a ligase detection reaction, a strand displacementamplification, a repair chain reaction, a cyclic probe reaction, a rapidamplification of cDNA ends, an invader assay, a bridge amplification orrolling circle amplification, or a combination thereof.

[0019] The present invention also provides methods for analyzing geneexpression including the steps of obtaining cDNA from multiple samples;amplifying a plurality of target sequences from the cDNA, therebyproducing a multiplex of amplification products; separating anddetecting the amplification products using a high throughput platform,wherein detecting generates a set of gene expression data; storing theset of gene expression data in a database; and performing a comparativeanalysis of the set of gene expression data.

[0020] The methods of the present invention optionally includeperforming one or more of the amplifying, separating or detecting stepsin a high throughput format. For example, the reactions can be performedin multi-well plates. Optionally, anywhere between about 96 and about5000 reactions, preferably between about 500 and 2000 reactions, andmore preferably about 1000 reactions, are performed per hour using themethods of the present invention. Furthermore, one or more miniaturizedscale platforms can be used to perform the methods of the presentinvention.

[0021] The present invention also provides systems for analyzing geneexpression. The elements of the system include, but are not limited to,a) an amplification module for producing a plurality of amplificationproducts from a pool of target sequences; b) a detection module fordetecting one or more members of the plurality of amplification productsand generating a set of gene expression data comprising a plurality ofdata points; and c) an analyzing module in operational communicationwith the detection module, the analyzing module comprising a computer orcomputer-readable medium comprising one or more logical instructionswhich organize the plurality of data points into a database and one ormore logical instructions which analyze the plurality of data points.Any or all of these modules can comprise high throughput technologiesand/or systems.

[0022] The amplification module of the present invention includes atleast one pair of universal primers and at least one pair oftarget-specific primers for use in the amplification process.Optionally, the amplification module includes a unique pair of universalprimers for each target sequence. Furthermore, the amplification modulecan include components to perform one or more of the followingreactions: a polymerase chain reaction, a transcription-basedamplification, a self-sustained sequence replication, a nucleic acidsequence based amplification, a ligase chain reaction, a ligasedetection reaction, a strand displacement amplification, a repair chainreaction, a cyclic probe reaction, a rapid amplification of cDNA ends,an invader assay, or various solution phase and/or solid phase assays(for example, bridge amplification or rolling circle amplification). Thedetection module can include systems for implementing separation of theamplification products; exemplary detection modules include, but are notlimited to, mass spectrometry instrumentation and electrophoreticdevices.

[0023] The analyzing module of the system includes one or more logicalinstructions for analyzing the plurality of data points generated by thedetection system. For example, the instructions can include software forperforming difference analysis upon the plurality of data points.Additionally (or alternatively), the instructions can include or beembodied in software for generating a graphical representation of theplurality of data points. Optionally, the instructions can be embodiedin system software which performs combinatorial analysis on theplurality of data points.

[0024] The present invention also provides kits for obtaining amultiplex set of amplification products of target genes andreferences-genes. The kits of the present invention include a) at leastone pair of universal primers; b) at least one pair of target-specificprimers; c) at least one pair of reference gene-specific primers; and d)one or more amplification reaction enzymes, reagents, or buffers. Thekits optionally further include software for storing and analyzing dataobtained from the amplification reactions.

[0025] Additionally, the present invention provides compositions forpreparing a plurality of amplification products from a plurality of mRNAtarget sequences. The compositions include one or more pairs ofuniversal primers; and one or more pairs of target-specific primers. Thepresent invention also provides for the use of the kits of the presentinvention for practicing any of the methods of the present invention, aswell as the use of a composition or kit as provided by the presentinvention for practicing a method of the present invention. Furthermore,the present invention provides assays utilizing any of these uses.

BRIEF DESCRIPTION ON THE FIGURES

[0026] The following drawings form part of the present specification andare included to further demonstrate certain aspects of the presentinvention. The invention may be better understood by reference to one ormore of these drawings in combination with the detailed description ofspecific embodiments presented herein.

[0027]FIG. 1: Schematic of one embodiment of a set of target-specificprimers and a universal primer employed in the present invention. Theabbreviation “TSP” indicates a target-specific primer, while “UP”indicates a universal primer. Different line patterns (bold, dashed,etc.) symbolize different DNA sequences.

[0028]FIG. 2: Schematic drawing depicting coupled target-specific anduniversal priming of a PCR reaction.

[0029]FIG. 3: Schematic depiction of exemplary reactions occurring in amultiplexed reverse transcriptase-based polymerase chain reaction(RT-PCR) reaction, using a combination of target-specific and universalprimers.

[0030]FIG. 4: Exemplary profiles of original and “shifted” multiplexgene sets.

[0031]FIG. 5: Exemplary profiles of multiplex gene sets using multiplefluorescent dye labels.

DETAILED DISCUSSION

[0032] Definitions

[0033] Before describing the present invention in detail, it is to beunderstood that this invention is not limited to particular compositionsor biological systems, which can, of course, vary. It is also to beunderstood that the terminology used herein is for the purpose ofdescribing particular embodiments only, and is not intended to belimiting. As used in this specification and the appended claims, thesingular forms “a”, “an” and “the” include plural referents unless thecontent clearly dictates otherwise. Thus, for example, reference to “adevice” includes a combination of two or more such devices, reference to“a gene fusion construct” includes mixtures of constructs, and the like.

[0034] Unless defined otherwise, all technical and scientific terms usedherein have the same meaning as commonly understood by one of ordinaryskill in the art to which the invention pertains. Although any methodsand materials similar or equivalent to those described herein can beused in the practice for testing of the present invention, currentlypreferred materials and methods are described herein.

[0035] In describing and claiming the present invention, the followingterminology will be used in accordance with the definitions set outbelow.

[0036] The term “absolute abundance” or “absolute gene expressionlevels” refers to the amount of a particular species (e.g., geneexpression product) present in a sample.

[0037] The term “amplified product” refers to a nucleic acid generatedby any method of nucleic acid amplification.

[0038] The term “attenuation” refers to a method of reducing the signalintensities of extremely abundant reaction products in a multiplex, suchthat the signals from all products of a multiplex set of products fallwithin the dynamic range of the detection platform used for the assay.

[0039] The term “blocking group” refers to a chemical modification atthe 3′ end of an amplification primer that does not interfere withhybridization between the primer and its target sequence, but cannot beextended by a DNA polymerase.

[0040] The term “cDNA” refers to complementary or “copy” DNA. GenerallycDNA is synthesized by a DNA polymerase using any type of RNA molecule(e.g., typically mRNA) as a template. Alternatively, the cDNA can beobtained by directed chemical syntheses.

[0041] The term “chemical treatment” refers to the process of exposing acell, cell line, tissue or organism to a chemical or biochemicalcompound (or library of compounds) that has/have the potential to alterits gene expression profile.

[0042] The term “complementary” refers to nucleic acid sequences capableof base-pairing according to the standard Watson-Crick complementaryrules, or being capable of hybridizing to a particular nucleic acidsegment under relatively stringent conditions. Nucleic acid polymers areoptionally complementary across only portions of their entire sequences.

[0043] The term “environmental stress” refers to an externally appliedfactor or condition that may cause an alteration in the gene expressionprofile of a cell.

[0044] The term “friction group” refers to a chemical or physical moietyattached to a nucleic acid for the purposes of reducing the mobility byfrictional drag of that nucleic acid in a matrix or fluid across whichan electric field is applied.

[0045] The term “gene” refers to a nucleic acid sequence encoding a geneproduct. The gene optionally comprises sequence information required forexpression of the gene (e.g., promoters, enhancers, etc.).

[0046] The term “gene expression” refers to transcription of a gene intoan RNA product, and optionally to translation into one or morepolypeptide sequences.

[0047] The term “gene expression data” refers to one or more sets ofdata that contain information regarding different aspects of geneexpression. The data set optionally includes information regarding: thepresence of target-transcripts in cell or cell-derived samples; therelative and absolute abundance levels of target transcripts; theability of various treatments to induce expression of specific genes;and the ability of various treatments to change expression of specificgenes to different levels.

[0048] The term “high throughput format” refers to analyzing more thanabout 10 samples per hour, preferably about 50 or more samples per hour,more preferably about 100 or more samples per hour, most preferablyabout 250, about 500, about 1000 or more samples per hour.

[0049] The term “hybridization” refers to duplex formation between twoor more polynucleotides, e.g., to form a double-stranded nucleic acid.The ability of two regions of complementarity to hybridize and remaintogether depends of the length and continuity of the complementaryregions, and the stringency of hybridization conditions.

[0050] The term “label” refers to any detectable moiety. A label may beused to distinguish a particular nucleic acid from others that areunlabeled, or labeled differently, or the label may be used to enhancedetection.

[0051] The terms “microplate,” “culture plate,” and “multiwell plate”interchangeably refer to a surface having multiple chambers, receptaclesor containers and generally used to perform a large number of discreetreactions simultaneously.

[0052] The term “miniaturized format” refers to procedures or methodsconducted at submicroliter volumes, including on both microfluidic andnanofluidic platforms.

[0053] The term “multiplex reaction” refers to a plurality of reactionsconducted simultaneously in a single reaction mixture.

[0054] The term “multiplex amplification” refers to a plurality ofamplification reactions conducted simultaneously in a single reactionmixture.

[0055] The term “nucleic acid” refers to a polymer of ribonucleic acidsor deoxyribonucleic acids, including RNA, mRNA, rRNA, tRNA, smallnuclear RNAs, cDNA, DNA, PNA, or RNA/DNA copolymers. Nucleic acid may beobtained from a cellular extract, genomic or extragenomic DNA, viral RNAor DNA, or artificially/chemically synthesized molecules.

[0056] The term “platform” refers to the instrumentation method used forsample preparation, amplification, product separation, productdetection, or analysis of data obtained from samples.

[0057] The term “primer” refers to any nucleic acid that is capable ofhybridizing at its 3′ end to a complementary nucleic acid molecule, andthat provides a free 3′ hydroxyl terminus which can be extended by anucleic acid polymerase.

[0058] The term “reference sequence” refers to a nucleic acid sequenceserving as a target of amplification in a sample that provides a controlfor the assay. The reference may be internal (or endogenous) to thesample source, or it may be an externally added (or exogenous) to thesample. An external reference may be either RNA, added to the sampleprior to reverse transcription, or DNA (e.g., cDNA), added prior to PCRamplification.

[0059] The term “relative abundance” or “relative gene expressionlevels” refers to the abundance of a given species relative to that of asecond species. Optionally, the second species is a reference sequence.

[0060] The term “RNA” refers to a polymer of ribonucleic acids,including RNA, mRNA, rRNA, tRNA, and small nuclear RNAs, as well as toRNAs that comprise ribonucleotide analogues to natural ribonucleic acidresidues, such as 2-O-methylated residues.

[0061] The term “semi-universal primer” refers to a primer that iscapable of hybridizing with more than one, but not all, of thetarget-specific primers in a multiplexed reaction.

[0062] The term “separation system” refers to any of a set ofmethodologies that can be employed to effect a size separation of theproducts of a reaction.

[0063] The term “size separation” refers to physical separation of acomplex mixture of species into individual components according to thesize of each species.

[0064] The term “target,” “target sequence,” or “target gene sequence”refers to a specific nucleic acid sequence, the presence, absence orabundance of which is to be determined. In a preferred embodiment of theinvention, it is a unique sequence within the mRNA of an expressed gene.

[0065] The term “target-specific primer” refers to a primer capable ofhybridizing with its corresponding target sequence. Under appropriateconditions, the hybridized primer can prime the replication of thetarget sequence.

[0066] The term “template” refers to any nucleic acid polymer that canserve as a sequence that can be copied into a complementary sequence bythe action of, for example, a polymerase enzyme.

[0067] The term “transcription” refers to the process of copying a DNAsequence of a gene into an RNA product, generally conducted by aDNA-directed RNA polymerase using the DNA as a template.

[0068] The term “treatment” refers to the process of subjecting one ormore cells, cell lines, tissues, or organisms to a condition, substance,or agent (or combinations thereof) that may cause the cell, cell line,tissue or organism to alter its gene expression profile. A treatment mayinclude a range of chemical concentrations and exposure times, andreplicate samples may be generated.

[0069] The term “universal primer” refers to a replication primercomprising a universal sequence.

[0070] The term “universal sequence” refers to a sequence contained in aplurality of primers, but preferably not in a complement to the originaltemplate nucleic acid (e.g., the target sequence), such that a primercomposed entirely of universal sequence is not capable of hybridizingwith the template.

[0071] Gene Expression as a Measure of the Biological State of a Cell

[0072] Transcription of genes into RNA is a critical early step in geneexpression. Consequently, the coordinated activation or suppression oftranscription of particular genes is an important component of theoverall regulation of expression. A variety of well-developed techniqueshave been established that provide ways to analyze and quantitate genetranscription.

[0073] Some of the earliest methods are based on detection of a label inRNA hybrids or protection of RNA from enzymatic degradation (see, forexample, Current Protocols in Molecular Biology, F. M. Ausubel et al.,eds., Current Protocols, a joint venture between Greene PublishingAssociates, Inc. and John Wiley & Sons, Inc., supplemented through1999). Methods based on detecting hybrids include northern blots andslot/dot blots. These two techniques differ in that the components ofthe sample being analyzed are resolved by size in a northern blot priorto detection, which enables identification of more than one speciessimultaneously. Slot blots are generally carried out using unresolvedmixtures or sequences, but can be easily performed in serial dilution,enabling a more quantitative analysis. Both techniques are verytime-consuming and require a fair amount of manual manipulation, makingthem expensive and unsuitable for high throughput applications.

[0074] In situ hybridization is a technique that monitors transcriptionby directly visualizing RNA hybrids in the context of a whole cell. Thismethod provides information regarding subcellular localization oftranscripts. However, it is not very quantitative, and is extremelytechnically demanding and time-consuming. As a consequence, thistechnique is best suited for basic research applications.

[0075] Techniques to monitor RNA that make use of protection fromenzymatic degradation include S1 analysis and RNAse protection assays(RPAs). Both of these assays employ a labeled nucleic acid probe, whichis hybridized to the RNA species being analyzed, followed by enzymaticdegradation of single-stranded regions of the probe. Analysis of theamount and length of probe protected from degradation is used todetermine the quantity and endpoints of the transcripts being studied.Although both methods can yield quantitative results, they aretime-consuming and cumbersome, making them poor candidates for ahigh-throughput, low cost general assay for gene expression.

[0076] A second family of assays developed for monitoring transcriptionmakes use of cDNA derived from mRNA. Because the material analyzed isDNA, these assays are less sensitive to degradation, and also providepartial and/or full clones with which to localize and clone genes orcoding sequences of interest. Methods include sequencing cDNA inserts ofan expressed sequence tag (EST) clone library (Adams et al. (1991)Science 252:1651-1656), which may be coupled with subtractivehybridization to improve sensitivity (Sagerstrom et al. (1997) AnnulRev. Biochem. 66:751-783), and serial analysis of gene expression(“SAGE”, described in U.S. Pat. No. 5,866,330 to Kinzler et al.;Velculescu et al. (1995) Science 270:484-487); and Zhang et al. (1997)Science 276:1268-1272). Both of these methods have been useful foridentification of novel, differentially expressed genes. However, theirmethodologies yield untargeted information, i.e., they survey the wholespectrum of mRNA in a sample rather than focusing on a predeterminedset. As a result, very large data sets are required to derive reliablequantitative data, making these methods inappropriate and far too costlyfor high throughput screening strategies.

[0077] Reverse transcriptase-mediated PCR (RT-PCR) gene expressionassays are directed at specified target gene products, overcoming someof the shortcomings described above. These assays are derivatives of PCRin which amplification is preceded by reverse transcription of mRNA intocDNA. Because the mRNA is amplified, this type of assay can detecttranscripts of very low abundance; however, the assay is notquantitative. Adaptations of this assay, called competitive RT-PCR(Becker-Andre and Hahlbrock (1989) Nucleic Acids Res. 17:9437-9446; Wanget al. (1989) Proc. Natl. Acad. Sci. USA 86:9717-9721; Gilliland et al.(1990) Proc. Natl. Acad. Sci. USA 87:2725-2729) have been developed thatare more quantitative. In these assays, a known amount of exogenoustemplate is added to the reaction mixture, to compete with the targetfor amplification. The exogenous competitor is titrated against thetarget, allowing for quantitation of a specified cDNA in the sample bycomparing the amplification of both templates within the same reactionmixture. Because titration is required to generate quantitative data,multiple reactions are required for each analysis. While this type ofassay is very sensitive and quantitative, these assays require multiplesteps in development, execution, and analysis, making them verytime-consuming, cumbersome, and expensive. The need to perform atitration reduces the overall throughput of the assay, and therequirement for an internal competitor for each target reduces themultiplexing capacity. These limitations restrict the usefulness of thisassay in analysis of large numbers of gene sets.

[0078] In order to increase the throughput of the RT-PCR assay, Su etal. (BioTechniques (1997) 22:1107-1113) combined microplate-based RNAextraction with multiplexed RT-PCR. With this method, they demonstratedsimultaneous analysis of three different target mRNAs amplified fromsamples prepared from a 96 well microplate. However, changes in geneexpression were only presented qualitatively.

[0079] Other methods for targeted mRNA analysis include differentialdisplay reverse transcriptase PCR (DDRT-PCR) and RNA arbitrarily primedPCR (RAP-PCR) (see U.S. Pat. No. 5,599,672; Liang and Pardee (1992)Science 257:967-971; Welsh et al. (1992) Nucleic Acids Res.20:4965-4970). Both methods use random priming to generate RT-PCRfingerprint profiles of transcripts in an unfractionated RNApreparation. The signal generated in these types of analyses is apattern of bands separated on a sequencing gel. Differentially expressedgenes appear as changes in the fingerprint profiles between two samples,which can be loaded in separate wells of the same gel. This type ofreadout allows identification of both up- and down-regulation of genesin the same reaction, appearing as either an increase or decrease inintensity of a band from one sample to another. However, due to thecomplexity of the fingerprint profile, amplification products arestrongly biased towards more abundant transcripts. Simultaneousamplification of hundreds to thousands of different productsdramatically compresses the dynamic range of measurement. The combinedresult of amplification bias, dynamic range compression and other biasesthat result from the use of a complex mix of primers eliminates theability to quantitate relative changes in expression between thedifferent genes in a sample. Furthermore, the methodology is designedfor identification of changes in the transcriptional profile of a wholecell, but does not provide any information about the identities of thePCR products. To identify a species, a band must be excised from thegel, subcloned, sequenced, and finally matched to a gene in a sequencedatabase. The complexity of the profile prohibits complete resolution ofPCR products on the gel, causing a high incidence of false positivesarising from multiple species existing in the same region of the gel.These characteristics make general fingerprinting techniques unsuitablefor investigation of already identified transcripts, and precludes ahigh-throughput quantitative analysis.

[0080] The TaqMan assay (Livak et al. (1995) PCR Methods Appl.4:357-362) is a quenched fluorescent dye system for quantitatingtargeted mRNA levels in a complex mixture. The assay has goodsensitivity and dynamic range, and yields quantitative results. Butbecause detection is based on fluorescence of unfractionated products,it can be multiplexed only to the very low levels (i.e., two to four) asallowed by resolution of emission spectra of the chromaphores.Furthermore, due to overlapping emission spectra, multiplexing reducesthe accuracy of quantitation. This limitation makes differentialanalysis problematic and increases the cost. Also, the assay isperformed in real time during thermal cycling, greatly reducing thethroughput of the assay.

[0081] Nucleic acid microarrays have been developed recently, which havethe benefit of assaying for sample hybridization to a large number ofprobes in a highly parallel fashion. They can be used for quantitationof mRNA expression levels, and dramatically surpass the above mentionedtechniques in terms of multiplexing capability. These arrays compriseshort DNA sequences, PCR products, or mRNA isolates fixed onto a solidsurface, which can then be used in a hybridization reaction with atarget sample, generally a whole cell extract (see, for example, U.S.Pat. Nos. 5,143,854 and 5,807,522; Fodor et al. (1991) Science251:767-773; and Schena et al. (1995) Science 270:467-470). Microarrayscan be used to measure the expression levels of several thousands ofgenes simultaneously, generating a gene expression profile of the entiregenome of relatively simple organisms. Each reaction, however, isperformed with a single sample against a very large number of geneprobes. As a consequence, microarray technology does not facilitate highthroughput analysis of very large numbers of unique samples against anarray of known probes.

[0082] The present invention addresses the need for gene expressiondetection and quantitation methodologies by providing novel methods foranalyzing gene expression, systems for implementing these techniques,compositions for preparing a plurality of amplification products from aplurality of mRNA target sequences, and related pools of amplificationproducts. The methods of the present invention include the steps of (a)obtaining a plurality of target cDNA sequences; (b) multiplex amplifyingthe target sequences using a plurality of target-specific primers andone or more universal primers; (c) separating one or more members of theresulting plurality of amplification products; (d) detecting the one ormore members of the plurality of amplification products, therebygenerating a set of gene expression data; (e) storing the data in adatabase; and (f) performing a comparative analysis on the set of geneexpression data, thereby analyzing the gene expression. The methods ofthe invention are highly sensitive; have a wide dynamic range; are rapidand inexpensive; have a high throughput; and allow the simultaneousdifferential analysis of a defined set of genes. The methods,compositions and kits of the invention also provide tools for geneexpression data collection and relational data analysis.

[0083] Methods for Quantitating Gene Expression Levels

[0084] The controlled expression of particular genes or groups of genesin a cell is the molecular basis for regulation of biological processesand, ultimately, for the physiological or pathological state of thecell. Knowledge of the “expression profile” of a cell is of keyimportance for answering many biological questions, including the natureand mechanism of cellular changes, or the degree of differentiation of acell, organ, or organism. Furthermore, the factors involved indetermining the expression profile may lead to the discovery of curesthat could reverse an adverse pathological or physiological condition. Adefined set of genes can be demonstrated to serve as indicators of aparticular state of a cell, and can therefore serve as a model formonitoring the cellular profile of gene expression in that state.

[0085] The pharmaceutical drug discovery process has traditionally beendominated by biochemical and enzymatic studies of a designated pathway.Although this approach has been productive, it is very laborious andtime-consuming, and is generally targeted to a single gene or definedpathway. Molecular biology and the development of gene cloning havedramatically expanded the number of genes that are potential drugtargets, and this process is accelerating rapidly as a result of theprogress made in sequencing the human genome. In addition to the growingset of available genes, techniques such as the synthesis ofcombinatorial chemical libraries have created daunting numbers ofcandidate drugs for screening. In order to capitalize on these availablematerials, methods are needed that are capable of extremely fast andinexpensive analysis of gene expression levels.

[0086] The present invention provides novel methods for the analysis ofchanges in expression levels of a set of genes. These methods includeproviding a plurality of target sequences, which are then analyzedsimultaneously in a multiplexed reaction. Multiplexing the analysisimproves the accuracy of quantitation; for example, signals from one ormore target genes can be compared to an internal control. Multiplexingalso reduces the time and cost required for analysis. Thus, the methodsof the present invention provide for rapid generation of a differentialexpression profile of a defined set of genes, through the comparison ofdata from multiple reactions.

[0087] The methods of the present invention include the steps of (a)obtaining a plurality of target nucleic acid sequences, generally cDNAsequences; (b) multiplex amplifying the target sequences using aplurality of target-specific primers and one or more universal primers;(c) separating one or more members of the resulting plurality ofamplification products; (d) detecting the one or more members of theplurality of amplification products, thereby generating a set of geneexpression data; (e) storing the data in a database; and (f) performinga comparative analysis on one or more components of the set of geneexpression data, thereby analyzing the gene expression. In analternative embodiment, the methods of the present invention include thesteps of obtaining cDNA from a plurality of samples for a plurality oftarget sequences; performing a plurality of multiplexed amplificationsof the target sequences, thereby producing a plurality of multiplexedamplification products; pooling the plurality of multiplexedamplification products; separating the plurality of multiplexedamplification products; detecting the plurality of multiplexedamplification products, thereby generating a set of gene expressiondata; storing the set of gene expression data in a database; andperforming a comparative analysis of the set of gene expression data. Inyet another embodiment, the methods of the present invention include thesteps of (a) obtaining cDNA from multiple samples; (b) amplifying aplurality of target sequences from the cDNA, thereby producing amultiplex of amplification products; (c) separating and detecting theamplification products using a high throughput platform, whereindetecting generates a set of gene expression data; (d) storing the setof gene expression data in a database; and (e) performing a comparativeanalysis of the set of gene expression data. In a further embodiment,the present invention provides methods for analyzing gene expression,including the steps of (a) obtaining cells, e.g. culturing one ofseveral designated cell lines; (b) optionally subjecting a set of thecultures to a specified treatment; (c) lysing the cells and isolatingone or more RNA molecules; (d) synthesizing cDNA first strand moleculesfrom a designated set of the mRNA molecules; (e) quantitativelyamplifying the resulting set of cDNA products using target-specificprimers in early rounds, coupled with amplifying the whole set byuniversal primers that have partial homology with all of thetarget-specific primers, and that contain a detectable label, preferablya fluorescent chromaphore, on at least one of the primers; (f)optionally pooling products of two or more separate reactions; (g)physically separating amplified products according to their length; (h)detecting and quantitating the separated amplification products, forexample, by deconvolution of data from any species of the same length(arising from reactions that were pooled); (i) determining the relativeabundance levels using an internal reference target; (j) storing theinformation in a gene expression database; and (k) performing acomparative analysis of the expression patterns. Each aspect of thesemethods of the present invention is addressed in greater detail below.

[0088] Sources of Target Sequences

[0089] Target sequences for use in the methods of the present inventionare obtained from a number of sources. For example, the target sequencescan be derived from organisms or from cultured cell lines. Cell typesutilized in the present invention can be either prokaryotic oreukaryotic cell types and/or organisms, including, but not limited to,animal cells, plants, yeast, fungi, bacteria, viruses, and the like.Target sequences can also be obtained from other sources, for example,needle aspirants or tissue samples from an organism (including, but notlimited to, mammals such as mice, rodents, guinea pigs, rabbits, dogs,cats, primates and humans; or non-mammalian animals such as nematodes,frogs, amphibians, various fishes such as the zebra fish, and otherspecies of scientific interest), non-viable organic samples or theirderivatives (such as a cell extract or a purified biological sample), orenvironmental sources, such as an air or water sample. Furthermore,target sequences can also be commercially or synthetically prepared,such as a chemical, phage, or plasmid library. DNA and/or RNA sequencesare available from a number of commercial sources, including The MidlandCertified Reagent Company (mcrc@oligos.com), The Great American GeneCompany (http://www.genco.com), ExpressGen Inc. (www.expressgen.com),Operon Technologies Inc. (Alameda, Calif.) and many others.

[0090] Cell lines which can be used in the methods of the presentinvention include, but are not limited to, those available from cellrepositories such as the American Type Culture Collection(www.atcc.org), the World Data Center on Microorganisms(http://wdcm.nig.ac.jp), European Collection of Animal Cell Culture(www.ecacc.org) and the Japanese Cancer Research Resources Bank(http://cellbank.nihs.go.jp). These cell lines include, but are notlimited to, the following cell lines: 293, 293Tet-Off, CHO-AA8 Tet-Off,MCF7, MCF7 Tet-Off, LNCap, T-5, BSC-1, BHK-21, Phinx-A, 3T3, HeLa, PC3,DU145, ZR 75-1, HS 578-T, DBT, Bos, CV1, L-2, RK13, HTTA, HepG2,BHK-Jurkat, Daudi, RAMOS, KG-1, K562, U937, HSB-2, HL-60, MDAHB231,C2C12, HTB-26, HTB-129, HPIC5, A-431, CRL-1573, 3T3L1, Cama-1, J774A.1,HeLa 229, PT-67, Cos7, OST7, HeLa-S, THP-1, and NXA. Additional celllines for use in the methods and matrices of the present invention canbe obtained, for example, from cell line providers such as CloneticsCorporation (Walkersville, Md.; www.clonetics.com). Optionally, theplurality of target sequences are derived from cultured cells optimizedfor the analysis of a particular disease area of interest, e.g., cancer,inflammation, cardiovascular disease, diabetes, infectious diseases,proliferative diseases, an immune system disorder, or a central nervoussystem disorder.

[0091] A variety of cell culture media are described in The Handbook ofMicrobiological Media, Atlas and Parks (eds) (1993, CRC Press, BocaRaton, Fla.). References describing the techniques involved in bacterialand animal cell culture include Sambrook et al., Molecular Cloning—ALaboratory Manual (2nd Ed.), Vol. 1-3 (1989, Cold Spring HarborLaboratory, Cold Spring Harbor, N.Y.); Current Protocols in MolecularBiology, F. M. Ausubel et al., eds., Current Protocols, (a joint venturebetween Greene Publishing Associates, Inc. and John Wiley & Sons, Inc.,supplemented through 2000); Freshney, Culture of Animal Cells, a Manualof Basic Technique, third edition (1994, Wiley-Liss, New York) and thereferences cited therein; Humason, Animal Tissue Techniques, fourthedition (1979, W. H. Freeman and Company, New York); and Ricciardelli,et al. (1989) In Vitro Cell Dev. Biol. 25:1016-1024. Informationregarding plant cell culture can be found in Plant Cell and TissueCulture in Liquid Systems, by Payne et al. (1992, John Wiley & Sons,Inc. New York, N.Y.); Plant Cell, Tissue and Organ Culture: FundamentalMethods by Gamborg and Phillips, eds. (1995, Springer Lab Manual,Springer-Verlag, Berlin), and is also available in commercial literaturesuch as the Life Science Research Cell Culture Catalogue (1998) fromSigma-Aldrich, Inc (St Louis, Mo.) (Sigma-LSRCCC) and the Plant CultureCatalogue and supplement (1997) also from Sigma-Aldrich, Inc (St Louis,Mo.) (Sigma-PCCS).

[0092] In an exemplary embodiment of methods of the present invention,either primary or immortalized (or other) cell lines are grown in amaster flask, then trypsinized (if they are adherent) and transferred toa 96-well plate, seeding each well at a density of 10⁴ to 10⁶cells/well. If the gene expression profile in response to a chemicaltreatment is sought, the chemical agent of choice is prepared in a rangeof concentrations. After a time of recovery and growth as appropriate tothe cell line, cells are exposed to the chemical for a period of timethat will not adversely impact the viability of the cells. Preferably,assays include a range of chemical concentrations and exposure times,and would include replicate samples. After treatment, medium is removedand cells are immediately lysed.

[0093] In further embodiments of cell culture, formats other than a96-well plate may be used. Other multiwell or microplate formatscontaining various numbers of wells, such as 6, 12, 48, 384, 1536 wells,or greater, are also contemplated. Culture formats that do not useconventional flasks, as well as microtiter formats, may also be used.

[0094] Treatment of Cells

[0095] The cells lines or sources containing the target nucleic acidsequences, are optionally subjected to one or more specific treatments,or in the case of organisms, may already be in different pathological orphysiological stages that induce changes in gene expression. Forexample, a cell or cell line can be treated with or exposed to one ormore chemical or biochemical constituents, e.g., pharmaceuticals,pollutants, DNA damaging agents, oxidative stress-inducing agents,pH-altering agents, membrane-disrupting agents, metabolic blockingagent; a chemical inhibitors, cell surface receptor ligands, antibodies,transcription promoters/enhancers/inhibitors, translationpromoters/enhancers/inhibitors, protein-stabilizing or destabilizingagents, various toxins, carcinogens or teratogens, characterized oruncharacterized chemical libraries, proteins, lipids, or nucleic acids.Optionally, the treatment comprises an environmental stress, such as achange in one or more environmental parameters including, but notlimited to, temperature (e.g. heat shock or cold shock), humidity,oxygen concentration (e.g., hypoxia), radiation exposure, culture mediumcomposition, or growth saturation. Alternatively, cultured cells may beexposed to other viable organisms, such as pathogens or other cells, tostudy changes in gene-expression that result from biological events,such as infections or cell-cell interactions. Responses to thesetreatments may be followed temporally, and the treatment can be imposedfor various times and at various concentrations. Target sequences canalso be derived from cells or organisms exposed to multiple specifictreatments as described above, either concurrently or in tandem (i.e., acancerous tissue sample may be further exposed to a DNA damaging agentwhile grown in an altered medium composition).

[0096] RNA Isolation

[0097] In some embodiments of the present invention, total RNA isisolated from samples for use as target sequences. Cellular samples arelysed once culture with or without the treatment is complete by, forexample, removing growth medium and adding a guanidinium-based lysisbuffer containing several components to stabilize the RNA. In someembodiments of the present invention, the lysis buffer also containspurified RNAs as controls to monitor recovery and stability of RNA fromcell cultures. Examples of such purified RNA templates include theKanamycin Positive Control RNA from Promega (Madison, Wis.), and 7.5 kbPoly(A)-Tailed RNA from Life Technologies (Rockville, Md.). Lysates maybe used immediately or stored frozen at, e.g., −80° C.

[0098] Optionally, total RNA is purified from cell lysates (or othertypes of samples) using silica-based isolation in anautomation-compatible, 96-well format, such as the Rneasy® purificationplatform (Qiagen, Inc.; Valencia, Calif.). Alternatively, RNA isisolated using solid-phase oligo-dT capture using oligo-dT bound tomicrobeads or cellulose columns. This method has the added advantage ofisolating mRNA from genomic DNA and total RNA, and allowing transfer ofthe mRNA-capture medium directly into the reverse transcriptasereaction. Other RNA isolation methods are contemplated, such asextraction with silica-coated beads or guanidinium. Further methods forRNA isolation and preparation can be devised by one skilled in the art.

[0099] Alternatively, the methods of the present invention are performedusing crude cell lysates, eliminating the need to isolate RNA. RNAseinhibitors are optionally added to the crude samples. When using crudecellular lysates, genomic DNA could contribute one or more copies oftarget sequence, depending on the sample. In situations in which thetarget sequence is derived from one or more highly expressed genes, thesignal arising from genomic DNA may not be significant. But for genesexpressed at very low levels, the background can be eliminated bytreating the samples with DNAse, or by using primers that target splicejunctions. For example, one of the two target-specific primers could bedesigned to span a splice junction, thus excluding DNA as a template. Asanother example, the two target-specific primers are designed to flank asplice junction, generating larger PCR products for DNA or unsplicedmRNA templates as compared to processed mRNA templates. One skilled inthe art could design a variety of specialized priming applications thatwould facilitate use of crude extracts as samples for the purposes ofthis invention.

[0100] Primer Design and Multiplex Strategies

[0101] Multiplex amplification of the target sequence involves combiningthe plurality of target sequences with a plurality of target-specificprimers and one or more universal primers, to produce a plurality ofamplification products. A multiplex set of target sequences optionallycomprises between about two targets and about 100 targets. In oneembodiment of the present invention, the multiplex reaction includes atleast 5 target sequences, but preferably at least ten targets or atleast fifteen targets. Multiplexes of much larger numbers (e.g., about20, about 50, about 75 and greater) are also contemplated.

[0102] In one embodiment of the methods of the present invention, atleast one of the amplification targets in the multiplex set is atranscript that is endogenous to the sample and has been independentlyshown to exhibit a fairly constant expression level (for example, a“housekeeping” gene). The signal from this endogenous reference sequenceprovides a control for converting signals of other gene targets intorelative expression levels. Optionally, a plurality of control mRNAtargets/reference sequences that have relatively constant expressionlevels may be included in the multiplexed amplification to serve ascontrols for each other. Alternatively, a defined quantity of anexogenous purified RNA species is added to the multiplex reaction or tothe cells, for example, with the lysis reagents. Almost any purified,intact RNA species can be used, e.g. the Kanamycin Positive Control RNAor the 7.5 kb Poly(A)-Tailed RNA mentioned previously. Thisexogenously-added amplification target provides a way to monitor therecovery and stability of RNA from cell cultures. It can also serve asan exogenous reference signal for converting the signals obtained fromthe sample mRNAs into relative expression levels. In still anotherembodiment, a defined quantity of a purified DNA species is added to thePCR to provide an exogenous reference target for converting the signalsobtained from sample mRNA targets into relative expression levels.

[0103] In one embodiment of the present invention, once the targets thatcomprise a multiplex set are determined, primer pairs complementary toeach target sequence are designed, including both target-specific anduniversal primers. This can be accomplished using any of severalsoftware products that design primer sequences, such as OLIGO (MolecularBiology Insights, Inc., CO), Gene Runner (Hastings Software Inc., NY),or Primer3 (The Whitehead Institute, MA). FIG. 1 illustrates theelements of design of exemplary target-specific primers (TSPs) anduniversal primers (UPs). Target specific primers (TSP1, TSP2, TSP3, TSP4and TSP5) are comprised of at least two portions. One portion, shown asa solid line within the 5′ region of each of the five TSP sequences,includes a region complementary to a selected “universal sequence.” Theuniversal sequence is utilized to allow amplification of multipletargets (having divergent sequences) while using the same primer (e.g.,the UP). The universal sequence is contained only in the primers, andpreferably is not present in any nucleic acid (or complement thereof)provided by the sample being tested. A second portion of the TSPs, shownas variable lines (solid, dotted, dashed, etc) within the 3′ region ofthe sequence, represents the sequence that is complementary to and willhybridize with one of a plurality of designated target sequences In FIG.1, a single universal primer (labeled as “UP”) is depicted; however,multiple universal primers having different or unique sequences orlabels can be employed in the methods of the present invention.Optionally, the primer design also includes consideration of propertiesbeyond the encoded sequence of the primer, such as annealingtemperature, 3′-end hybridization stability, and minimization ofsequences that would allow annealing among the primers themselves.

[0104] Oligonucleotide primers are typically prepared by thephosphoramidite approach. In this automated, solid-phase procedure, eachnucleotide is individually added to the 5′-end of the growingoligonucleotide chain, which is in turn attached at the 3′-end to asolid support. The added nucleotides are in the form of trivalent3′-phosphoramidites that are protected from polymerization by adimethoxytrityl (“DMT”) group at the 5′-position. After base inducedphosphoramidite coupling, mild oxidation to give a pentavalentphosphotriester intermediate and DMT removal provides a new site foroligonucleotide elongation. These syntheses may be performed on, forexample, a Perkin Elmer/Applied Biosystems Division DNA synthesizer. Theoligonucleotide primers are then cleaved off the solid support, and thephosphodiester and exocyclic amino groups are deprotected with ammoniumhydroxide.

[0105] Nucleic Acid Hybridization

[0106] The length of complementary sequence between each primer and itsbinding partner (i.e. the target sequence or the universal sequence)should be sufficient to allow hybridization of the primer only to itstarget within a complex sample at the annealing temperature used for thePCR. A complementary sequence of, for example, about 15, 16, 17, 18, 19,20, 21, 22, 23, 24, or 25 or more nucleotides is preferred for both thetarget-specific and universal regions of the primers. A particularlypreferred length of each complementary region is about 20 bases, whichwill promote formation of stable and specific hybrids between the primerand target.

[0107] Nucleic acids “hybridize” when they associate, typically insolution. Nucleic acids hybridize due to a variety of well characterizedphysico-chemical forces, such as hydrogen bonding, solvent exclusion,base stacking and the like. An extensive guide to the hybridization ofnucleic acids is found in Tijssen (1993) Laboratory Techniques inBiochemistry and Molecular Biology—Hybridization with Nucleic AcidProbes, part I, chapter 2, “Overview of principles of hybridization andthe strategy of nucleic acid probe assays,” (Elsevier, N.Y.), as well asin Ausubel, supra. Hames and Higgins (1995) Gene Probes 1, IRL Press atOxford University Press, Oxford, England (Hames and Higgins 1) and Hamesand Higgins (1995) Gene Probes 2, IRL Press at Oxford University Press,Oxford, England (Hames and Higgins 2) provide details on the synthesis,labeling, detection and quantification of DNA and RNA, includingoligonucleotides.

[0108] “Stringent hybridization wash conditions” in the context ofnucleic acid hybridization experiments, such as Southern and northernhybridizations, are sequence dependent, and are different underdifferent environmental parameters. An extensive guide to thehybridization of nucleic acids is found in Tijssen (1993), supra, and inHames and Higgins 1 and Hames and Higgins 2, supra.

[0109] For purposes of the present invention, generally, “highlystringent” hybridization and wash conditions are selected to be about 5°C. or less lower than the thermal melting point (T_(m)) for the specificsequence at a defined ionic strength and pH (as noted below, highlystringent conditions can also be referred to in comparative terms). TheT_(m) is the temperature (under defined ionic strength and pH) at which50% of the test sequence hybridizes to a perfectly matched primer. Verystringent conditions are selected to be equal to the T_(m) for aparticular primer.

[0110] The T_(m) is the temperature of the nucleic acid duplexesindicates the temperature at which the duplex is 50% denatured under thegiven conditions and its represents a direct measure of the stability ofthe nucleic acid hybrid. Thus, the T_(m) corresponds to the temperaturecorresponding to the midpoint in transition from helix to random coil;it depends on length, nucleotide composition, and ionic strength forlong stretches of nucleotides.

[0111] After hybridization, unhybridized nucleic acid material can beremoved by a series of washes, the stringency of which can be adjusteddepending upon the desired results. Low stringency washing conditions(e.g., using higher salt and lower temperature) increase sensitivity,but can product nonspecific hybridization signals and high backgroundsignals. Higher stringency conditions (e.g., using lower salt and highertemperature that is closer to the hybridization temperature) lowers thebackground signal, typically with only the specific signal remaining.See, Rapley, R. and Walker, J. M. eds., Molecular Biomethods Handbook(Humana Press, Inc. 1998) (hereinafter “Rapley and Walker”), which isincorporated herein by reference in its entirety for all purposes.

[0112] Thus, one measure of stringent hybridization is the ability ofthe primer to hybridize to one or more of the target nucleic acids (orcomplementary polynucleotide sequences thereof) under highly stringentconditions. Stringent hybridization and wash conditions can easily bedetermined empirically for any test nucleic acid.

[0113] For example, in determining highly stringent hybridization andwash conditions, the hybridization and wash conditions are graduallyincreased (e.g., by increasing temperature, decreasing saltconcentration, increasing detergent concentration and/or increasing theconcentration of organic solvents, such as formalin, in thehybridization or wash), until a selected set of criteria are met. Forexample, the hybridization and wash conditions are gradually increaseduntil a target nucleic acid, and complementary polynucleotide sequencesthereof, binds to a perfectly matched complementary nucleic acid.

[0114] A target nucleic acid is said to specifically hybridize to aprimer nucleic acid when it hybridizes at least ½ as well to the primeras to a perfectly matched complementary target, i.e., with a signal tonoise ratio at least ½ as high as hybridization of the primer to thetarget under conditions in which the perfectly matched primer binds tothe perfectly matched complementary target with a signal to noise ratiothat is at least about 2.5×-10×, typically 5×-10× as high as thatobserved for hybridization to any of the unmatched target nucleic acids.

[0115] Optionally, primers are designed such that the annealingtemperature of the universal sequence is higher/greater than that of thetarget-specific sequences. Method employing these primers furtherinclude increasing the annealing temperature of the reaction after thefirst few rounds of amplification. This increase in reaction temperaturesuppresses further amplification of sample nucleic acids by the TSPs,and drives amplification by the UP. Depending on the applicationenvisioned, one skilled in the art can employ varying conditions ofhybridization to achieve varying degrees of selectivity of primertowards the target sequence. For example, varying the stringency ofhybridization or the position of primer hybridization can revealdivergence within gene families.

[0116] Optionally, each candidate primer is shown or proven to becompatible with the other primers used in a multiplex reaction. In apreferred embodiment, each target-specific primer pair produces a singleamplification product of a predicted size from a sample minimallycontaining all of the targets of the multiplex, and more preferably froma crude RNA mixture. Preferably, amplification of each individual targetby its corresponding primers is not inhibited by inclusion of any otherprimers in the multiplex. None of the primers, either individually or incombination, should produce spurious products. These issues are easilyaddressed by one of skill in the art without the need for excessiveundue experimentation.

[0117] Inherent Properties and Labels

[0118] Primer sequences are optionally designed to accommodate one ormore detection techniques that can be employed while performing themethods of the present invention. For example, detection of theamplification products is optionally based upon one or more inherentproperties of the amplification products themselves, such as mass ormobility. Other embodiments utilize methods of detection based onmonitoring a label associated with the PCR products. In theseembodiments, generally one or more of the universal primers contains thelabel. Optionally, the label is a fluorescent chromaphore. A fluorescentlabel may be covalently attached, noncovalently intercalated, or may bean energy transfer label. Other useful labels include mass labels, whichare incorporated into amplification products and released after thereaction for detection, chemiluminescent labels, electrochemical andinfrared labels, isotopic derivatives, nanocrystals, or any of variousenzyme-linked or substrate-linked labels detected by the appropriateenzymatic reaction.

[0119] One preferred embodiment of the methods of the present inventionincludes the use and detection of one or more fluorescent labels.Generally, fluorescent molecules each display a distinct emissionspectrum, thereby allowing one to employ a plurality of fluorescentlabels in a multiplexed reaction, and then separate the mixed data intoits component signals by spectral deconvolution. Exemplary fluorescentlabels for use in the methods of the present invention include a singledye covalently attached to the molecule being detected, a single dyenoncovalently intercalated into product DNA, or an energy-transferfluorescent label.

[0120] Other embodiments of labeling include mass labels, which areincorporated into amplification products and released after the reactionfor detection; chemiluminescent, electrochemical, and infrared labels;radioactive isotopes; and any of various enzyme-linked orsubstrate-linked labels detectable by the appropriate enzymaticreaction. Many other useful labels are known in the art, and one skilledin the art can envision additional strategies for labeling amplificationproducts of the present invention.

[0121] Cleavable Linkages and Size-Shifting of Amplification Products

[0122] Primers can also be designed to produce amplification productshaving sizes which can selectively be changed, or “shifted” afteramplification, in order to better resolve the amplification productsprior to or during detection. For example, a primer can be designed toincorporate a restriction enzyme site within a portion of the amplifiedproduct. The products of this reaction can then optionally be cleavedenzymatically to generate size-shifted amplification products.Alternatively, primers can be designed to incorporate variouschemically-cleavable linkages, mass labels, or other linkers which canoptionally be used in the detection of one or more of the amplificationproducts.

[0123] Linking groups, or linkers, can also be incorporated into theprimers of the present invention. Linking groups of use in the presentinvention can have a range of structures, substituents and substitutionpatterns. They can, for example be derivitized with nitrogen, oxygenand/or sulfur containing groups which are pendent from, or integral to,the linker group backbone. Examples include, polyethers, polyacids(polyacrylic acid, polylactic acid), polyols (e.g., glycerol,),polyamines (e.g., spermine, spermidine) and molecules having more thanone nitrogen, oxygen and/or sulfur moiety (e.g., 1,3-diamino-2-propanol,taurine). See, for example, Sandier et al. Organic Functional GroupPreparations 2nd Ed., Academic Press, Inc. San Diego 1983.

[0124] Methods for preparing linkers that can be incorporated intoprimers for use in the methods of the present invention are known in theart. Numerous linking groups compatible with phosphoramidite chemistryare commercially available (Glen Research, Sterling, Va.) and canreadily be incorporate into oligonucleotides during automated synthesisprocedures.

[0125] One of skill will recognize that a linker that is appropriate forincorporation into a nucleic acid oligomer synthesis can also beutilized to derivatize a nucleic acid monomer. For example, chemicallycleavable primers can be used in the amplification step of the methodsof the present invention. In these embodiments, one or more of theprimers used in amplification contain a chemical linkage, such as athiophosphate moiety, that can be selectively cleaved, generating twoseparate fragments from the primer. Cleavage is optionally performedafter the amplification reaction, e.g., by removing a fixed number ofnucleotides from the 5′ end of products made from that primer. Designand use of such primers is described in detail in, for example, Li et al(Electrophoresis (1999) 20:1258-1265), PCT publication WO 96/37630(Monforte et al.) and U.S. Pat. No. 5,700,642 (Monforte et al.) and U.S.Pat. No. 6,090,558 (Butler et al.), which are incorporated herein byreference in their entirety for all purposes.

[0126] Exemplary Primer Designs for Use in a Multiplexed AmplificationReaction

[0127] A preferred embodiment of the invention utilizes a combination ofTSPs that will hybridize with one of a plurality of designated targetsequences, and universal primers (UPs) for amplification of multipletargets in the multiplexed reaction. Optionally, the primary way ofseparating the signals of the multiplexed amplification is according toproduct sizes. Alternatively, the signals can be resolved usingdifferential labeling to separate signals from products of similar size.To separate products according to size, the predicted sizes must beconsidered in primer design. FIG. 1 illustrates the elements of designof these primers. Each of the TSPs has a universal sequence within the5′ region, which is shared among the primers, but not contained in theoriginal template (i.e. the target sequence). This universal sequencemay be the same or different for the forward and reverse TSPs. Followingthe 3′ end of the universal sequence is a target-specific sequence forannealing to and amplifying the target sequence (e.g., gene) ofinterest.

[0128] The universal primer is composed of the universal sequence heldin common within the 5′ regions of the TSPs. If a single UP is to beused, the universal sequence will be the same within all TSPs. If a UPpair is to be used, the universal sequence will be different in theforward and reverse primers of the TSPs. The UP may also contain adetectable label on at least one of the primers, such as a fluorescentchromaphore. Both the target-specific and universal sequences are ofsufficient length and sequence complexity to form stable and specificduplexes, allowing amplification and detection of the target gene.

[0129] Elimination of Variations in Primer Annealing Efficiency

[0130] Variations in primer length and sequence can also have a largeimpact on the efficiency with which primers anneal to their target andprime replication. In a typical multiplexed reaction in which eachproduct is amplified by a unique primer pair, the relative quantities ofamplified products may be significantly altered from the relativequantities of targets due to difference in annealing efficiencies.Embodiments of the methods of the present invention that couple the useof target-specific primers and universal primers eliminates this bias,producing amplification products that accurately reflect relative mRNAlevels.

[0131] Coupled Target-Specific and Universal Priming of the PCR

[0132] In the methods of the present invention, the amounts of eachdesignated target are amplified to improve the sensitivity and dynamicrange of the assay. In some embodiments to monitor gene expression,cellular RNA is isolated and reverse transcribed to obtain cDNA, whichis then used as template for amplification. In other embodiments, cDNAmay be provided and used directly. The primers described for use in thepresent invention can be used in any one of a number oftemplate-dependent processes that amplify sequences of the target geneand/or its expressed transcripts present in a given sample. Other typesof templates may also be used, such as tRNA, rRNA, or othertranscription products, genomic DNA, viral nucleic acids, and syntheticnucleic acid polymers. Several methods described below are contemplated.

[0133] A preferred embodiment of the methods of the present inventionemploys PCR, which is described in detail in U.S. Pat. No. 4,683,195(Mullis et al.), U.S. Pat. No. 4,683,202 (Mullis), and U.S. Pat. No.4,800,159 (Mullis et al.), and in PCR Protocols A Guide to Methods andApplications (Innis et al., eds.) Academic Press Inc. San Diego, Calif.(1990). PCR utilizes pairs of primers having sequences complimentary toopposite strands of target nucleic acids, and positioned such that theprimers are converging. The primers are incubated with template DNAunder conditions that permit selective hybridization. Primers may beprovided in double-stranded or single-stranded form, although thesingle-stranded form is preferred. If the target gene(s) sequence ispresent in a sample, the primers will hybridize to form anucleic-acid:primer complex. An excess of deoxynucleoside triphosphatesis added, along with a thermostable DNA polymerase, e.g. Taq polymerase.If the target gene(s):primer complex has been formed, the polymerasewill extend the primer along the target gene(s) sequence by addingnucleotides. After polymerization, the newly-synthesized strand of DNAis dissociated from its complimentary template strand by raising thetemperature of the reaction mixture. When the temperature issubsequently lowered, new primers will bind to each of these two strandsof DNA, and the process is repeated. Multiple cycles of raising andlowering the temperature are conducted, with a round of replication ineach cycle, until a sufficient amount of amplification product isproduced.

[0134]FIG. 2 illustrates the TSP-UP coupled priming strategy. Heavierlines represent a DNA template; thinner lines depict the oligonucleotideprimers. Primer nomenclature is as described in the legend to FIG. 1.The lower case “f” and “r” in the primer names indicate a forward orreverse orientation. Lines “A,” “B,” “C,” and “D” represent uniquenucleic acid sequences, and “A′,” “B′,” “C′,” and “D′” indicate theirrespective complementary sequences. “B” and “C” sequences derive fromthe template; “A” and “D” sequences derive from universal primersequences. Arrowheads indicate directionality. A vertical bar indicatesan endpoint of the DNA strand. The first set of reactions (first arrow)occur in the early PCR cycles (for example, in only the first and secondPCR cycles); in these reaction, primarily the TSPs are used as primers,and the resulting products will have UP sequences added to both ends,flanking the amplified target sequence. The second set of reactions(second, reiterative arrow) occur in all subsequent PCR cycles; both TSPand UP primers are used, but the UPs dominate when present in molarexcess over the TSPs.

[0135] In early rounds of the amplification, replication is primedprimarily by the TSPs. The first round will add the universal sequenceto the 5′ regions of the amplification products. The second cycle willgenerate sequence complementary to the universal sequence within the 3′region of the complementary strand, creating a template that can beamplified by the universal primers alone. Optionally, the reaction isdesigned to contain limiting amounts of each of the TSPs and a molarexcess of the UP, such that the UP will generally prime replication onceits complementary sequence has been established in the template. Themolar excess of UP over a TSP can range from about 5:1 to about 100: 1;optionally, the reaction utilizes approximately 10:1 molar excess of UPover the amount of each TSP. Because all of the TSPs contain the sameuniversal sequence, the same universal primer will amplify all targetsin the multiplex, eliminating the quantitative variation that resultsfrom amplification from different primers.

[0136] Amplification Methods

[0137] In a preferred embodiment of the methods of the presentinvention, RNA is converted to cDNA using a target-specific primercomplementary to the RNA for each gene target being monitored in themultiplex set in a reverse-transcription (RT) reaction. Methods ofreverse transcribing RNA into cDNA are well known, and described inSambrook, supra. Alternative methods for reverse transcription utilizethermostable DNA polymerases, as described in the art. As an exemplaryembodiment, avian myeloblastosis virus reverse transcriptase (AMV-RT),or Maloney murine leukemia virus reverse transcriptase (MoMLV-RT) isused, although other enzymes are contemplated. An advantage of usingtarget-specific primers in the RT reaction is that only the desiredsequences are converted into a PCR template. No superfluous primers orcDNA products are carried into the subsequent PCR amplification.

[0138] In another embodiment of the amplifying step, RNA targets arereverse transcribed using non-specific primers, such as an anchoredoligo-dT primer, or random sequence primers. An advantage of thisembodiment is that the “unfractionated” quality of the mRNA sample ismaintained because the sites of priming are non-specific, i.e., theproducts of this RT reaction will serve as template for any desiredtarget in the subsequent PCR amplification. This allows samples to bearchived in the form of DNA, which is more stable than RNA.

[0139] In other embodiments of the methods of the present invention,transcription-based amplification systems (TAS) are used, such as thatfirst described by Kwoh et al. (Proc. Natl. Acad. Sci. (1989)86(4):1173-7), or isothermal transcription-based systems such as 3SR(Self-Sustained Sequence Replication; Guatelli et al. (1990) Proc. Natl.Acad. Sci. 87:1874-1878) or NASBA (nucleic acid sequence basedamplification; Kievits et al. (1991) J Virol Methods. 35(3):273-86). Inthese methods, the mRNA target of interest is copied into cDNA by areverse transcriptase. The primer for cDNA synthesis includes thepromoter sequence of a designated DNA-dependent RNA polymerase 5′ to theprimer's region of homology with the template. The resulting cDNAproducts can then serve as templates for multiple rounds oftranscription by the appropriate RNA polymerase. Transcription of thecDNA template rapidly amplifies the signal from the original targetmRNA. The isothermal reactions bypass the need for denaturing cDNAstrands from their RNA templates by including RNAse H to degrade RNAhybridized to DNA.

[0140] In other embodiments, amplification is accomplished by used ofthe ligase chain reaction (LCR), disclosed in European PatentApplication No. 320,308 (Backman and Wang), or by the ligase detectionreaction (LDR), disclosed in U.S. Pat. No. 4,883,750 (Whiteley et al.).In LCR, two probe pairs are prepared, which are complimentary eachother, and to adjacent sequences on both strands of the target. Eachpair will bind to opposite strands of the target such that they abut.Each of the two probe pairs can then be linked to form a single unit,using a thermostable ligase. By temperature cycling, as in PCR, boundligated units dissociate from the target, then both molecules can serveas “target sequences” for ligation of excess probe pairs, providing foran exponential amplification. The LDR is very similar to LCR. In thisvariation, oligonucleotides complimentary to only one strand of thetarget are used, resulting in a linear amplification of ligationproducts, since only the original target DNA can serve as ahybridization template. It is used following a PCR amplification of thetarget in order to increase signal.

[0141] In further embodiments, several methods generally known in theart would be suitable methods of amplification. Some additional examplesinclude, but are not limited to, strand displacement amplification(Walker et al. (1992) Nucleic Acids Res. 20:1691-1696), repair chainreaction (REF), cyclic probe reaction (REF), solid-phase amplification,including bridge amplification (Mehta and Singh (1999) BioTechniques26(6): 1082-1086), rolling circle amplification (Kool, U.S. Pat. No.5,714,320), rapid amplification of cDNA ends (Frohman (1988) Proc. Natl.Acad. Sci. 85: 8998-9002), and the “invader assay” (Griffin et al.(1999) Proc. Natl. Acad. Sci. 96: 6301-6306).

[0142] Attenuation of Strong Signals

[0143] The set of targets included in a multiplex reaction generally allyield signal strengths within the dynamic range of the detectionplatform used in order for quantitation of gene expression to beaccurate. In some embodiments, it may be desirable or necessary toinclude a very highly expressed gene in a multiplex assay. However, thehighly-expressed gene can impact the accuracy of quantitation for othergenes expressed at very low levels if its signal is not attenuated. Themethods of the current invention provide ways for attenuating thesignals of relatively abundant targets during the amplification reactionsuch that they can be included in a multiplexed set without impactingthe accuracy of quantitation of that set.

[0144] Toward this end, amplification primers are optionally used thatblock polymerase extension of the 3′ end of the primer. One preferredembodiment is modification of the 3′-hydroxyl of the oligonucleotideprimer by addition of a phosphate group. Another preferred embodiment isattachment of the terminal nucleotide via a 3′-3′ linkage. One skilledin the art can conceive of other chemical structures or modificationsthat can be used for this purpose. The modified and the correspondingunmodified primer for the highly abundant target are mixed in a ratioempirically determined to reduce that target's signal, such that itfalls within the dynamic range of other targets of the multiplex.Preferably, the reverse target-specific primer is modified, therebyattenuating signal by reduction of the amount of template created in thereverse transcriptase reaction.

[0145] Another embodiment for signal attenuation entails use of atarget-specific primer that contains the target-specific sequence, butno universal primer sequence. This abbreviated primer (sans universalsequence) and the corresponding primer containing the universal sequencewithin the 5′ region are mixed in a ratio empirically determined toreduce that target's signal, such that it then falls within the dynamicrange of other targets of the multiplex system.

[0146] Multiplex Amplification Strategies

[0147] An important embodiment of the methods of the present inventioninvolves the use of various PCR multiplexing strategies that are madepossible by the combined use of target-specific and universal primers.An illustration of the fundamental multiplexed reaction is shown in FIG.3.

[0148] The numbers 1 through 6 on the left represent six differentreactions occurring simultaneously in a single mixture. Column Arepresents the six target sequences of the multiplex. Column B depictsthe templates and primers in the PCR amplification. Lines shown asparallel and having opposite directionality represent complementarysequences. The templates are initially single-stranded mRNA molecules,but eventually are predominantly DNA amplification products that serveas template in subsequent cycles. Messenger RNA is converted to cDNA bythe action of reverse transcriptase polymerization from thetarget-specific reverse primers (TSPr1-6) for each of the six targets.The six target-specific forward primers (TSPf1-6) and the universalforward and reverse primers (UPf1-6, UPr1-6) are added along with athermostable polymerase to generate the second strand of cDNA, followedby PCR amplification. The drawings in Column B show single-strandedtemplates with the TSPs aligned (depicted as parallel) at their sites ofhybridization. The UP can anneal to target DNA only after itscomplementary universal sequence is added to the opposite strand throughreplication across the 5′ region of the TSP. Column C shows the productsof PCR amplification. Products contain the target sequences (TS1-6) thatwere the targets of amplification, flanked by the universal primersequences (UP) that were added to the ends of the target sequences bythe target-specific primers. The TSPf and TSPr primers are specific, soby definition they will all be unique. However, the two universalprimers may be the same sequence as each other or different sequences,i.e., the UPf may be the same sequence as the UPr. Furthermore, subsetsof target sequences in the multiplex set may be amplified by differentUPs, i.e., the UPf1-6 primers and/or UPr1-6 primers may be of one ormultiple sequences.

[0149] All of these examples are variations on the fundamental RT-PCRassay shown in FIG. 3. For the sake of simplicity, only strategies usingfluorescent dyes are illustrated, although many of the other labelingstrategies previously discussed could be applied.

[0150] Data Collection

[0151] The number of species than can be detected within a mixturedepends primarily on the resolution capabilities of the separationplatform used, and the detection methodology employed. A preferredembodiment of the separation step of the methods of the presentinvention is based upon size-based separation technologies. Onceseparated, individual species are detected and quantitated by eitherinherent physical characteristics of the molecules themselves, ordetection of a label associated with the DNA.

[0152] Embodiments employing other separation methods are alsodescribed. For example, certain types of labels allow resolution of twospecies of the same mass through deconvolution of the data. Non-sizebased differentiation methods (such as deconvolution of data fromoverlapping signals generated by two different fluorophores) allowpooling of a plurality of multiplexed reactions to further increasethroughput.

[0153] Optionally, the throughput rate for the detection step is betweenabout 100 and 5000 samples per hour, preferably between about 250 and2500 samples, and more preferably about 1000 samples per hour perseparation system (i.e., one mass spectrometer, one lane of a gel, orone capillary of a capillary electrophoresis device). In order tofurther reduce assay costs and increase the throughput of the overallprocess, sample-handling is optionally conducted in a miniaturizedformat. For the methods of the present invention, miniaturized formatsare those conducted at submicroliter volumes, including bothmicrofluidic and nanofluidic platforms. Any or all of the amplification,separation, and/or detection steps of the present can utilizeminiaturized formats and platforms. For example, many of the modes ofseparation described below are presently available in a miniaturizedscale.

[0154] Separation Methods

[0155] Preferred embodiments of the present invention incorporate a stepof separating the products of a reaction based on their sizedifferences. The PCR products generated during the multiplexamplification optionally range from about 50 to about 500 bases inlength, which can be resolve from one another by size. Any one ofseveral devices may be used for size separation, including massspectrometry, any of several electrophoretic devices, includingcapillary, polyacrylamide gel, or agarose gel electrophoresis, or any ofseveral chromatographic devices, including column chromatography, HPLC,or FPLC.

[0156] One preferred embodiment for sample analysis is massspectrometry. Several modes of separation that determine mass arepossible, including Time-of-Flight (TOF), Fourier Transform MassSpectrometry (FFMS), and quadruple mass spectrometry. Possible methodsof ionization include Matrix-Assisted Laser Desorption and Ionization(MALDI) or Electrospray Ionization (ESI). A preferred embodiment for theuses described in this invention is MALDI-TOF (Wu, et al. (1993) RapidCommunications in Mass Spectrometry 7: 142-146). This method may be usedto provide unfragmented mass spectra of mixed-base oligonucleotidescontaining between about 1 and about 1000 bases. In preparing the samplefor analysis, the analyte is mixed into a matrix of molecules thatresonantly absorb light at a specified wavelength. Pulsed laser light isthen used to desorb oligonucleotide molecules out of the absorbing solidmatrix, creating free, charged oligomers and minimizing fragmentation.The preferred solid matrix material for this purpose is3-hydroxypicolinic acid (Wu, supra), although others are contemplated.

[0157] In another preferred embodiment, the device of the invention is amicrocapillary for analysis of nucleic acids obtained from the sample.Microcapillary electrophoresis generally involves the use of a thincapillary or channel, which may optionally be filled with a particularmedium to improve separation, and employs an electric field to separatecomponents of the mixture as the sample travels through the capillary.Samples composed of linear polymers of a fixed charge-to-mass ratio,such as DNA, will separate based on size. The high surface to volumeratio of these capillaries allows application of very high electricfields across the capillary without substantial thermal variation,consequently allowing very rapid separations. When combined withconfocal imaging methods, these methods provide sensitivity in the rangeof attomoles, comparable to the sensitivity of radioactive sequencingmethods. The use of microcapillary electrophoresis in size separation ofnucleic acids has been reported in Woolley and Mathies (Proc. Natl.Acad. Sci. USA (1994) 91:11348-11352).

[0158] Capillaries are optionally fabricated from fused silica, oretched, machined, or molded into planar substrates. In manymicrocapillary electrophoresis methods, the capillaries are filled withan appropriate separation/sieving matrix. Several sieving matrices areknown in the art that may be used for this application, including, e.g.,hydroxyethyl cellulose, polyacrylamide, agarose, and the like.Generally, the specific gel matrix, running buffers and runningconditions are selected to obtain the separation required for aparticular application. Factors that are considered include, e.g., sizesof the nucleic acid fragments, level of resolution, or the presence ofundenatured nucleic acid molecules. For example, running buffers mayinclude agents such as urea to denature double-stranded nucleic acids ina sample.

[0159] Microfluidic systems for separating molecules such as DNA and RNAare commercially available and are optionally employed in the methods ofthe present invention. For example, the “Personal Laboratory System” andthe “High Throughput System” have been developed by CaliperTechnologies, Corp. (Mountain View, Calif.). The Agilent 2100, whichuses Caliper Technologies' LabChip™ microfluidic systems, is availablefrom Agilent Technologies (Palo Alto, Calif.). Currently, specializedmicrofluidic devices which provide for rapid separation and analysis ofboth DNA and RNA are available from Caliper Technologies for the Agilent2100. See, e.g., http://www.calipertech.com.

[0160] Other embodiments are generally known in the art for separatingPCR amplification products by electrophoresis through gel matrices.Examples include polyacrylamide, agarose-acrylamide, or agarose gelelectrophoresis, using standard methods (Sambrook, supra).

[0161] Alternatively, chromatographic techniques may be employed forresolving amplification products. Many types of physical or chemicalcharacteristics may be used to effect chromatographic separation in thepresent invention, including adsorption, partitioning (such as reversephase), ion-exchange, and size exclusion. Many specialized techniqueshave been developed for their application including methods utilizingliquid chromatography or HPLC (Katz and Dong (1990) BioTechniques8(5):546-55; Gaus et al. (1993) J. Immunol. Methods 158:229-236).

[0162] In yet another embodiment of the separation step of the presentinvention, cDNA products are captured by their affinity for certainsubstrates, or other incorporated binding properties. For example,labeled cDNA products such as biotin or antigen can be captured withbeads bearing avidin or antibody, respectively. Affinity capture isutilized on a solid support to enable physical separation. Many types ofsolid supports are known in the art that would be applicable to thepresent invention. Examples include beads (e.g. solid, porous,magnetic), surfaces (e.g. plates, dishes, wells, flasks, dipsticks,membranes), or chromatographic materials (e.g. fibers, gels, screens).

[0163] Certain separation embodiments entail the use of microfluidictechniques. Technologies include separation on a microcapillaryplatform, such as designed by ACLARA BioSciences Inc. (Mountain View,Calif.), or the LabChip™ microfluidic devices made by CaliperTechnologies Inc. Another recent technology developed by Nanogen, Inc.(San Diego, Calif.), utilizes microelectronics to move and concentratebiological molecules on a semiconductor microchip. The microfluidicsplatforms developed at Orchid Biosciences, Inc. (Princeton, N.J.),including the Chemtel™ Chip which provides for parallel processing ofhundreds of reactions, can be used in the present invention. Thesemicrofluidic platforms require only nanoliter sample volumes, incontrast to the microliter volumes required by other conventionalseparation technologies.

[0164] Fabrication of microfluidic devices, including microcapillaryelectrophoretic devices, has been discussed in detail, e.g., Regnier etal. (Trends Biotechnol. (1999) 17(3):101-6), Deyl et al. (Forensic Sci.Int. (1998) 92:89-124), Effenhauser et al. (Electrophoresis (1997)18:2203-2213), and U.S. Pat. No. 5,904,824 (Oh). Typically, the methodsmake use of photolithographic etching of micron-scale channels on asilica, silicon, or other crystalline substrate or chip. In someembodiments, capillary arrays may be fabricated using polymericmaterials with injection-molding techniques. These methods can bereadily adapted for use in miniaturized devices of the presentinvention.

[0165] Some of the processes usually involved in genetic analysis havebeen miniaturized using microfluidic devices. For example, PCTpublication WO 94/05414 reports an integrated micro-PCR apparatus forcollection and amplification of nucleic acids from a specimen. U.S. Pat.No. 5,304,487 (Wilding et al.) and U.S. Pat. No. 5,296,375 (Kricka etal.) discuss devices for collection and analysis of cell-containingsamples. U.S. Pat. No. 5,856,174 (Lipshutz et al.) describes anapparatus that combines the various processing and analytical operationsinvolved in nucleic acid analysis.

[0166] Additional technologies are also contemplated. For example,Kasianowicz et al. (Proc. Natl. Acad. Sci. USA (1996) 93:13770-13773)describe the use of ion channel pores in a lipid bilayer membrane fordetermining the length of polynucleotides. In this system, an electricfield is generated by the passage of ions through the pores.Polynucleotide lengths are measured as a transient decrease of ioniccurrent due to blockage of ions passing through the pores by the nucleicacid. The duration of the current decrease was shown to be proportionalto polymer length. Such a system can be applied as a size separationplatform in the present invention.

[0167] The target-specific primers and universal primers of the presentinvention are useful both as reagents for hybridization in solution,such as priming PCR amplification, as well as for embodiments employinga solid phase, such as microarrays. With microarrays, sample nucleicacids such as mRNA or DNA are fixed on a selected matrix or surface. PCRproducts may be attached to the solid surface via one of theamplification primers, then denatured to provide single-stranded DNA.This-spatially-partitioned, single-stranded nucleic acid is then subjectto hybridization with selected probes under conditions that allow aquantitative determination of target abundance. In this embodiment,amplification products from each individual multiplexed reaction are notphysically separated, but are differentiated by hybridizing with a setof probes that are differentially labeled. Alternatively, unextendedamplification primers may be physically immobilized at discreetpositions on the solid support, then hybridized with the products of amultiplexed PCR amplification for quantitation of distinct specieswithin the sample. In this embodiment, amplification products areseparated by way of hybridization with probes that are spatiallyseparated on the solid support.

[0168] Separation platforms may optionally be coupled to utilize twodifferent separation methodologies, thereby increasing the multiplexingcapacity of reactions beyond that which can be obtained by separation ina single dimension. For example, some of the RT-PCR primers of amultiplex reaction may be coupled with a moiety that allows affinitycapture, while other primers remain unmodified. Samples are then passedthrough an affinity chromatography column to separate PCR productsarising from these two classes of primers. Flow-through fractions arecollected and the bound fraction eluted. Each fraction may then befurther separated based on other criteria, such as size, to identifyindividual components.

[0169] The invention also includes rapid analytical method using one ormore microfluidic handling systems. For example, a subset of primers ina multiplex reaction would contain a hydrophobic group. Separation isthen performed in two dimensions, with hydrophilic partitioning in onedirection, followed by size separation in the second direction. The useof a combination of dyes can further increase the multiplex size.

[0170] Detection Methods

[0171] Following separation of the different products of the multiplex,one or more of the member species is detected and/or quantitated. Someembodiments of the methods of the present invention enable directdetection of products. Other embodiments detect reaction products via alabel associated with one or more of the amplification primers. Manytypes of labels suitable for use in the present invention are known inthe art, including chemiluminescent, isotopic, fluorescent,electrochemical, inferred, or mass labels, or enzyme tags. In furtherembodiments, separation and detection may be a multi-step process inwhich samples are fractionated according to more than one property ofthe products, and detected one or more stages during the separationprocess.

[0172] One embodiment of the invention requiring no labeling ormodification of the molecules being analyzed is detection of themass-to-charge ratio of the molecule itself. This detection technique isoptionally used when the separation platform is a mass spectrometer. Anembodiment for increasing resolution and throughput with mass detectionis in mass-modifying the amplification products. Nucleic acids can bemass-modified through either the amplification primer or thechain-elongating nucleoside triphosphates. Alternatively, the productmass can be shifted without modification of the individual nucleic acidcomponents, by instead varying the number of bases in the primers.Several types of moieties have been shown to be compatible with analysisby mass spectrometry, including polyethylene glycol, halogens, alkyl,aryl, or aralkyl moieties, peptides (described in, for example, U.S.Pat. No. 5,691,141). Isotopic variants of specified atoms, such asradioisotopes or stable, higher mass isotopes, are also used to vary themass of the amplification product. Radioisotopes can be detected basedon the energy released when they decay, and numerous applications oftheir use are generally known in the art. Stable (non-decaying) heavyisotopes can be detected based on the resulting shift in mass, and areuseful for distinguishing between two amplification products that wouldotherwise have similar or equal masses. Other embodiments of detectionthat make use of inherent properties of the molecule being analyzedinclude ultraviolet light absorption (UV) or electrochemical detection.Electrochemical detection is based on oxidation or reduction of achemical compound to which a voltage has been applied. Electrons areeither donated (oxidation) or accepted (reduction), which can bemonitored as current. For both UV absorption and electrochemicaldetection, sensitivity for each individual nucleotide varies dependingon the component base, but with molecules of sufficient length this biasis insignificant, and detection levels can be taken as a directreflection of overall nucleic acid content.

[0173] Several embodiments of the detecting step of the presentinvention are designed to identify molecules indirectly by detection ofan associated label. A number of labels may be employed that provide afluorescent signal for detection (see, for example, www.probes.com). Ifa sufficient quantity of a given species is generated in a reaction, andthe mode of detection has sufficient sensitivity, then some fluorescentmolecules may be incorporated into one or more of the primers used foramplification, generating a signal strength proportional to theconcentration of DNA molecules. Several fluorescent moieties, includingAlexa 350, Alexa 430, AMCA, BODIPY 630/650, BODIPY 650/665, BODIPY-FL,BODIPY-R6G, BODIPY-TMR, BODIPY-TRX, carboxyfluorescein, Cascade Blue,Cy3, Cy5, 6-FAM, Fluorescein, HEX, 6-JOE, Oregon Green 488, Oregon Green500, Oregon Green 514, Pacific Blue, REG, Rhodamine Green, RhodarmineRed, ROX, TAMRA, TET, Tetramethylrhodamine, and Texas Red, are generallyknown in the art and routinely used for identification of discreetnucleic acid species, such as in sequencing reactions. Many of thesedyes have emission spectra distinct from one another, enablingdeconvolution of data from incompletely resolved samples into individualsignals. This allows pooling of separate reactions that are each labeledwith a different dye, increasing the throughput during analysis, asdescribed in more detail below.

[0174] The signal strength obtained from fluorescent dyes can beenhanced through use of related compounds called energy transfer (ET)fluorescent dyes. After absorbing light, ET dyes have emission spectrathat allow them to serve as “donors” to a secondary “acceptor” dye thatwill absorb the emitted light and emit a lower energy fluorescentsignal. Use of these coupled-dye systems can significantly amplifyfluorescent signal. Examples of ET dyes include the ABI PRISM BigDyeterminators, recently commercialized by Perkin-Elmer Corporation (FosterCity, Calif.) for applications in nucleic acid analysis. Thesechromaphores incorporate the donor and acceptor dyes into a singlemolecule and an energy transfer linker couples a donor fluorescein to adichlororhodamine acceptor dye, and the complex is attached to a DNAreplication primer.

[0175] Fluorescent signals can also be generated by non-covalentintercalation of fluorescent dyes into nucleic acids after theirsynthesis and prior to separation. This type of signal will vary inintensity as a function of the length of the species being detected, andthus signal intensities must be normalized based on size. Severalapplicable dyes are known in the art, including, but not limited to,ethidium bromide and Vistra Green. Some intercalating dyes, such as YOYOor TOTO, bind so strongly that separate DNA molecules can each be boundwith a different dye and then pooled, and the dyes will not exchangebetween DNA species. This enables mixing separately generated reactionsin order to increase multiplexing during analysis.

[0176] Alternatively, technologies such as the use of nanocrystals as afluorescent DNA label (Alivisatos, et al. (1996) Nature 382:609-11) canbe employed in the methods of the present invention. Another method,described by Mazumder, et al. (Nucleic Acids Res. (1998) 26:1996-2000),describes hybridization of a labeled oligonucleotide probe to its targetwithout physical separation from unhybridized probe. In this method, theprobe is labeled with a chemiluminescent molecule that in the unboundform is destroyed by sodium sulfite treatment, but is protected inprobes that have hybridized to target sequence.

[0177] In another embodiment, products may be detected and quantitatedby monitoring a set of mass labels, each of which are specificallyassociated with one species of amplification reaction. The labels arereleased by either chemical or enzymatic mechanisms after theamplification reaction. Release is followed by size separation of themixture of labels to quantitate the amount of each species of theamplification reaction. Separation methods that can be employed includemass spectrometry, capillary electrophoresis, or HPLC. Such strategies,and their applications for detection of nucleic acids, have beendescribed in, for example, U.S. Pat. No. 6,104,028 (Hunter et al.) andU.S. Pat. No. 6,051,378 (Monforte et al.), as well as PCT publicationsWO 98/26095 (Monforte et al.) and WO 97/27327 (Van Ness et al.).

[0178] In further embodiments, both electrochemical and infrared methodsof detection can be amplified over the levels inherent to nucleic acidmolecules through attachment of EC or IR labels. Their characteristicsand use as labels are described in, for example, PCT publication WO97/27327. Some preferred compounds that can serve as an IR label includean aromatic nitrile, aromatic alkynes, or aromatic azides. Numerouscompounds can serve as an EC label; many are listed in PCT publicationWO 97/27327.

[0179] Enzyme-linked reactions are also employed in the detecting stepof the methods of the present invention. Enzyme-linked reactionstheoretically yield an infinite signal, due to amplification of thesignal by enzymatic activity. In this embodiment, an enzyme is linked toa secondary group that has a strong binding affinity to the molecule ofinterest. Following separation of the nucleic acid products, enzyme isbound via this affinity interaction. Nucleic acids are then detected bya chemical reaction catalyzed by the associated enzyme. Various couplingstrategies are possible utilizing well-characterized interactionsgenerally known in the art, such as those between biotin and avidin, anantibody and antigen, or a sugar and lectin. Various types of enzymescan be employed, generating colorimetric, fluorescent, chemiluminescent,phosphorescent, or other types of signals. As an illustration, a PCRprimer may be synthesized containing a biotin molecule. After PCRamplification, DNA products are separated by size, and those made withthe biotinylated primer are detected by binding with streptavidin thatis covalently coupled to an enzyme, such as alkaline phosphatase. Asubsequent chemical reaction is conducted, detecting bound enzyme bymonitoring the reaction product. The secondary affinity group may alsobe coupled to an enzymatic substrate, which is detected by incubationwith unbound enzyme. One of skill in the art can conceive of manypossible variations on the different embodiments of detection methodsdescribed above.

[0180] In some embodiments, it may be desirable prior to detection toseparate a subset of amplification products from other components in thereaction, including other products. Exploitation of known high-affinitybiological interactions can provide a mechanism for physical capture. Insome embodiments of this process, the 5′ region of one of the universalprimers contains a binding moiety that allows capture of the products ofthat primer. Some examples of high-affinity interactions include thosebetween a hormone with its receptor, a sugar with a lectin, avidin andbiotin, or an antigen with its antibody. After affinity capture,molecules are retrieved by cleavage, denaturation, or eluting with acompetitor for binding, and then detected as usual by monitoring anassociated label. In some embodiments, the binding interaction providingfor capture may also serve as the mechanism of detection.

[0181] Furthermore, the size of an amplification product or products areoptionally changed, or “shifted,” in order to better resolve theamplification products from other products prior to detection. Forexample, chemically cleavable primers can be used in the amplificationreaction. In this embodiment, one or more of the primers used inamplification contains a chemical linkage that can be broken, generatingtwo separate fragments from the primer. Cleavage is performed after theamplification reaction, removing a fixed number of nucleotides from the5′ end of products made from that primer. Design and use of such primersis described in detail in, for example, PCT publication WO 96/37630.

[0182] One preferred embodiment of the methods of the present inventionis the generation of gene expression profiles. However, several otherapplications are also possible, as would be apparent to one skilled inthe art from a reading of this disclosure. For example, the methods ofthe present invention can be used to investigate the profile andexpression levels of one or more members of complex gene families. As anillustration, cytochrome P-450 isozymes form a complex set of closelyrelated enzymes that are involved in detoxification of foreignsubstances in the liver. The various isozymes in this family have beenshown to be specific for different substrates. Design of target-specificprimers that anneal to variant regions in the genes provides an assay bywhich their relative levels of induction in response to drug treatmentscan be monitored. Other examples include monitoring expression levels ofalleles with allele-specific primers, or monitoring mRNA processing withprimers that specifically hybridize to a spliced or unspliced region, orto splice variants. One skilled in the art could envision otherapplications of the present invention that would provide a method tomonitor genetic variations or expression mechanisms.

[0183] Systems for Gene Expression Analysis

[0184] The present invention also provides systems for analyzing geneexpression. The elements of the system include, but are not limited to,an amplification module for producing a plurality of amplificationproducts from a pool of target sequences; a detection module fordetecting one or more members of the plurality of amplification productsand generating a set of gene expression data; and an analyzing modulefor organizing and/or analyzing the data points in the data set. Any orall of these modules can comprise high throughput technologies and/orsystems.

[0185] The amplification module of the system of the present inventionproduces a plurality of amplification products from a pool of targetsequences. The amplification module includes at least one pair ofuniversal primers and at least one pair of target-specific primers foruse in the amplification process. Optionally, the amplification moduleincludes a unique pair of universal primers for each target sequence.Furthermore, the amplification module can include components to performone or more of the following reactions: a polymerase chain reaction, atranscription-based amplification, a self-sustained sequencereplication, a nucleic acid sequence based amplification, a ligase chainreaction, a ligase detection reaction, a strand displacementamplification, a repair chain reaction, a cyclic probe reaction, a rapidamplification of cDNA ends, an invader assay, a bridge amplification, arolling circle amplification, solution phase and/or solid phaseamplifications, and the like.

[0186] The detection module detects the presence, absence, or quantityof one or more members of the plurality of amplification products.Additionally, the detection module generates a set of gene expressiondata, generally in the form of a plurality of data points. The detectionmodule optionally further comprises a separation module for separationof one or more members of the multiplexed reaction prior to, or during,operation of the detection module. The detection module, or the optionalseparation module, can include systems for implementing separation ofthe amplification products; exemplary detection modules include, but arenot limited to, mass spectrometry instrumentation and electrophoreticdevices.

[0187] The third component of the system of the present invention, theanalyzing module, is in operational communication with the detectionmodule. The analyzing module of the system includes, e.g., a computer orcomputer-readable medium having one or more one or more logicalinstructions for analyzing the plurality of data points generated by thedetection system. The analyzing system optionally comprises multiplelogical instructions; for example, the logical instructions can includeone or more instructions which organize the plurality of data pointsinto a database and one or more instructions which analyze the pluralityof data points. The instructions can include software for performingdifference analysis upon the plurality of data points. Additionally (oralternatively), the instructions can include or be embodied in softwarefor generating a graphical representation of the plurality of datapoints. Optionally, the instructions can be embodied in system softwarewhich performs combinatorial analysis on the plurality of data points.

[0188] The computer employed in the analyzing module of the presentinvention can be, e.g., a PC (Intel x86 or Pentium chip-compatible DOS™,OS2™ WINDOWS™ WINDOWS NT™, WINDOWS95™, WINDOWS98™, or WINDOWS ME™), aLINUX based machine, a MACINTOSH™, Power PC, or a UNIX based machine(e.g., SUN™ work station) or other commercially common computer which isknown to one of skill. Software for computational analysis is available,or can easily be constructed by one of skill using a standardprogramming language such as VisualBasic, Fortran, Basic, C, C++, Java,or the like. Standard desktop applications such as word processingsoftware (e.g., Microsoft Word™ or Corel WordPerfect™) and databasesoftware (e.g., spreadsheet software such as Microsoft Excel™, CorelQuattro Pro™, or database programs such as Microsoft Access™ orParadox™) can also be used in the analyzing system of the presentinvention.

[0189] The computer optionally includes a monitor that is often acathode ray tube (“CRT”) display, a flat panel display (e.g., activematrix liquid crystal display, liquid crystal display), or others.Computer circuitry is often placed in a box that includes numerousintegrated circuit chips, such as a microprocessor, memory, interfacecircuits, and others. The box also optionally includes a hard diskdrive, a floppy disk drive, a high capacity removable drive such as awriteable CD-ROM, and other common peripheral elements. Inputtingdevices such as a keyboard or mouse optionally provide for input from auser and for user selection of sequences to be compared or otherwisemanipulated in the relevant computer system.

[0190] The computer typically includes appropriate software forreceiving user instructions, either in the form of user input into a setparameter fields, e.g., in a GUI, or in the form of preprogrammedinstructions, e.g., preprogrammed for a variety of different specificoperations. The software then converts these instructions to appropriatelanguage for instructing the operation of the fluid direction andtransport controller to carry out the desired operation.

[0191] The software can also include output elements for displayingand/or further analyzing raw data, massaged data, or proposed resultsfrom one or more computational processes involved in the analysis of thegene expression data set.

[0192] Kits

[0193] In an additional aspect, the present invention provides kitsembodying the methods, compositions, and systems for analysis of geneexpression as described herein. Kits of the present invention optionallycomprise one or more of the following, preferably in a spatiallyseparate arrangement: a) at least one pair of universal primers; b) atleast one pair of target-specific primers; c) at least one pair ofreference gene-specific primers; and d) one or more amplificationreaction enzymes, reagents, or buffers. Optionally, the universalprimers provided in the kit include labeled primers, such as thosedescribed in the present application and the references cited herein.The target-specific primers can vary from kit to kit, depending upon thespecified target gene(s) to be investigated. Exemplary referencegene-specific primers (e.g., target-specific primers for directingtranscription of one or more reference genes) include, but are notlimited to, primers for β-actin, cyclophilin, GAPDH, and various rRNAmolecules.

[0194] The kits of the invention optionally include one or morepreselected primer sets that are specific for the genes to be amplified.The preselected primer sets optionally comprise one or more labelednucleic acid primers, contained in suitable receptacles or containers.Exemplary labels include, but are not limited to, a fluorophore, a dye,a radiolabel, an enzyme tag, etc., that is linked to a nucleic acidprimer itself.

[0195] In one embodiment, kits that are suitable for use in PCR areprovided. In PCR kits, target-specific and universal primers areprovided which include sequences that have sequences from, and hybridizeto spatially distinct regions of one or more target genes. Optionally,pairs of target-specific primers are provided. Generally, thetarget-specific primers are composed of at least two parts: a universalsequence within the 5′ portion that is complementary to a universalprimer sequence, and a sequence within the 3′ portion (and optionally,proximal to the universal sequence) for recognition of a target gene. Insome embodiments of the invention, the set of targets monitored in ananalysis may be specified by a client for use in a proprietary testingor screening application. In an alternate embodiment, standardizedtarget sets may be developed for general applications, and constitutecomponents of the kits described below. Kits of either of theseembodiment can be used to amplify all genes, unknown and/or known, thatrespond to certain treatments or stimuli.

[0196] In addition, one or more materials and/or reagents required forpreparing a biological sample for gene expression analysis areoptionally included in the kit. Furthermore, optionally included in thekits are one or more enzymes suitable for amplifying nucleic acids,including various polymerases (RT, Taq, etc.), one or moredeoxynucleotides, and buffers to provide the necessary reaction mixturefor amplification.

[0197] In one preferred embodiment of the invention, the kits areemployed for analyzing gene expression patterns using mRNA as thestarting template. The mRNA template may be presented as either totalcellular RNA or isolated mRNA; both types of sample yield comparableresults. In other embodiments, the methods and kits described in thepresent invention allow quantitation of other products of geneexpression, including tRNA, rRNA, or other transcription products. Instill further embodiments, other types of nucleic acids may serve astemplate in the assay, including genomic or extragenomic DNA, viral RNAor DNA, or nucleic acid polymers generated by non-replicative orartificial mechanism, including PNA or RNA/DNA copolymers.

[0198] Optionally, the kits of the present invention further includesoftware to expedite the generation, analysis and/or storage of data,and to facilitate access to databases. The software includes logicalinstructions, instructions sets, or suitable computer programs that canbe used in the collection, storage and/or analysis of the data.Comparative and relational analysis of the data is possible using thesoftware provided.

[0199] The kits optionally comprise distinct containers for eachindividual reagent and enzyme, as well as for each probe or primer pair.Each component will generally be suitable as aliquoted in its respectivecontainer. The container of the kits optionally includes at least onevial, ampule, or test tube. Flasks, bottles and other containermechanisms into which the reagents can be placed and/or aliquoted arealso possible. The individual containers of the kit are preferablymaintained in close confinement for commercial sale. Suitable largercontainers may include injection or blow-molded plastic containers intowhich the desired vials are retained. Instructions, such as writtendirections or videotaped demonstrations detailing the use of the kits ofthe present invention, are optionally provided with the kit.

[0200] In a further aspect, the present invention provides for the useof any composition or kit herein, for the practice of any method orassay herein, and/or for the use of any apparatus or kit to practice anyassay or method herein.

EXAMPLES

[0201] The methods of the present invention are particularly suited foranalyzing gene expression patterns. The present invention providesmethods for the rapid generation of a differential expression profile ofa defined set of genes through comparison of data from multiplereactions. Multiple differential expression profiles can be used forcomparison of different cell types, or of a single cell type exposed todifferent environmental conditions, or in various developmental ordisease states. The methods of the present invention provide a way togenerate large bodies of differential expression data, which can be usedfor modeling a matrix of gene product interactions for whole cells.Relational analysis is used with large and complex sets of geneexpression profiles, and is of valuable for identification of potentialtherapeutic targets, screening of candidate drugs, diagnostics, andother potential uses.

[0202] The methods of the present invention can also be suitablymodified for the analysis of other biological processes, including, butnot limited to, genotyping, mapping, mutation analysis, forensics, oranalysis of other RNA molecules such as tRNAs, rRNAs, or hnRNAs.

[0203] The following examples are included to demonstrate variousembodiments of the present invention. It will be appreciated by those ofskill in the art that the techniques disclosed in the examples whichfollow represent techniques determined by the inventor to function wellin the practice of the invention, and thus can be considered toconstitute preferred modes for its practice. However, those of skill inthe art should, in light of the present disclosure, appreciate that manychanges can be made in the specific embodiments which are disclosed andstill obtain a like or similar result without departing from the spiritand scope of the invention.

Example 1 Cell Culture and Chemical Exposure

[0204] The hepatocyte cell line, Hep G2 (human hepatocellular carcinoma,obtained from the American Type Culture Collection, Rockville Md.,ATCC#HB-8065), was used to evaluate the effects of various chemicals onexpression of a set of genes known to be involved in cellulartoxicological responses. The cells were routinely maintained in T75flasks in Eagle's MEM medium (with non-essential amino acids, sodiumpyruvate, and Earle's salts) and 10% fetal bovine serum at 37° C. in ahumidified atmosphere of 5% CO2. The chemicals used in exposureexperiments included cadmium chloride (CdCl2) and methyl methanesulfonate (MMS). CdCl2 is a strong inducer of metallothionein, ametal-binding protein, and is known to be carcinogenic and capable ofinterfering with DNA repair. MMS is an alkylating agent that induces DNAdamage. Dilutions of these compounds were prepared from concentratedstocks obtained from Aldrich Chemical Company (Milwaukee, Wis.). Waterwas used as the solvent control in dosing studies. Approximately 0.02 mLof a dilution of each toxin was added to 2 mL of culture medium, withfinal concentrations ranging from 10-4M to 10-6M CdCl2 and from 0.5 mMto 2 mM MMS. These concentration ranges were empirically determined tonot be lethal to cells for the duration of the exposure period. Toperform exposures, cells were trypsinized and transferred to twelve-welldishes, seeding each well at a density of 1×104 cells/well. After 4 daysof recovery and growth, cells were exposed to the designated toxin for 3hours. Medium was then removed and cells immediately lysed. Cell numberwas quantitated using a dye incorporation assay, CyQUANT from MolecularProbes (Eugene, Oreg.).

Example 2 RNA Isolation

[0205] Total RNA was purified from crude cell lysates using Rneasy®total RNA purification kits from Qiagen Inc. (Valencia, Calif.), in anautomation-compatible, 96-well format. In order to monitor recovery andstability of RNA from cell cultures, two purified RNA samples (KanamycinPositive Control RNA from Promega (Madison, Wis.), and 7.5 kbPoly(A)-Tailed RNA from Life Technologies (Rockville, Md.)) were addedwith the lysis reagents. After the cellular treatments were complete,growth medium was removed and cells were lysed under denaturingconditions with RLT buffer (Qiagen, Valencia, Calif.) containingguanidine isothiocyanate and beta-mercapto ethanol to inactivate RNAses.Ethanol was then added to promote binding of RNA to the RNeasy membrane,and the entire volumes of the samples were loaded into the wells of amultiwell plate. The silica gel membrane of the RNeasy kit specificallybinds total RNA, allowing contaminants to be washed away in flow-throughprocessing of the membrane using a vacuum manifold. Samples bound to themembrane were dried by centrifugation of the plate. In order to eluteRNA, 45 μL of RNAse-free water was added to each sample well, incubated,collected by centrifugation, and then the elution process repeated.Samples were stable in this form, and were stored at −80° C. for lateruse in expression assays.

Example 3 Reverse Transcription to Generate cDNA

[0206] A multiplex primer mix was designed to amplify ten target mRNAs,including four controls and six test targets. Two of the controls wereendogenous cellular mRNAs that exhibit constant expression levels(β-actin and cyclophilin), allowing for normalization of signals fromother genes. Two additional control RNA targets were added exogenouslyin the cell lysis buffer to provide a means to monitor recovery andstability of RNA from cell lysates (kanamycin mRNA and the 7.5 kb RNA aspreviously described). Six test genes were chosen that had been shown inprior art to exhibit changes in the amount of mRNA transcribed fromthose genes in response to a specific challenge.

[0207] Reverse transcription and PCR™ amplification primers weredesigned for the gene multiplex set using OLIGO 5.0 (Molecular BiologyInsights, Inc., Cascade, Colo.). The sizes of the predicted PCRamplification products of the nine targets ranged from 100 to 330 bases,with the smallest size difference being 5 bases. The length ofcomplementary sequence between each target-specific primer and itstarget sequence was 20 bases, and the length of complementary sequencebetween the target-specific primers and the universal primers was 18bases. Primers were synthesized by Operon Technologies Inc. (Alameda,Calif.), or by chemists at GeneTrace Systems Inc. (Alameda, Calif.),utilizing conventional phosphoramidite synthesis techniques.

[0208] A mixture of reverse target-specific primers appropriate for themultiplex was prepared and diluted to a working concentration of 0.02μM. (Reverse priming of β-actin mRNA is attenuated by addition of asecond, inhibitory reverse target-specific primer. See Example 4.) Tobegin the reverse transcription step, 30 ng of total RNA, prepared asdescribed in Example 2, was mixed with the reverse primers, 10 units ofMoloney Murine Leukemia Virus Reverse Transcriptase (MoMLV-RT, PromegaInc.), and deoxyribonucleotides (1 mM from Promega) in an appropriatebuffer (20 mM Tris HCl, 16.7 mM MgCl₂, pH 8.3, and 2.5 units RNasin).Samples were incubated at 42° C. for 30 minutes, followed by 95° C. for5 minutes to inactivate the enzyme.

Example 4 Signal Attenuation

[0209] If one of the targets in a multiplex set is present at very highlevels, it may be necessary to attenuate the signal generated by thattarget to ensure that all signals fall within the dynamic range of theassay. The β-actin mRNA provided one such example, as this mRNA isconstitutively expressed at very high levels. Amplification of theβ-actin signal was attenuated by using a mixture of two target-specificreverse primers, the first terminating at the 3′ end with a hydroxylgroup which is extendible by a reverse transcriptase, and the secondcontaining a phosphate group attached to the 3′-hydroxyl which blocksextension by reverse transcriptase. The blocked 13-actin primer was usedin a 40-fold excess relative to the extendible primer, and the combinedconcentration was equivalent to the concentrations of all othertarget-specific reverse primers in the multiplex. This amount ofinhibition typically resulted in about a 70% reduction in conversion ofmRNA to cDNA.

Example 5 Multiplex Amplification of Target Sequences Using a SingleUnlabeled Universal Primer

[0210] After inactivation of the reverse transcriptase, the cDNAproducts were used directly as templates in a PCR amplification. Amixture of forward target-specific primers appropriate for the multiplexreaction was prepared (SEQ ID No. 1-22). A single unlabeled universalprimer was used for amplification; both the forward and reversetarget-specific primers in the multiplex composition were designed tocontain the same universal sequence within their 5′ regions. The forwardtarget-specific primers and the universal primer were diluted to aworking concentration of 10 nM and 500 nM respectively, and then addedto the samples from the reverse transcriptase reaction, along with 1unit TaqGOLD® (Perkin-Elmer Applied Biosystems Inc., Foster City,Calif.) and 375 μM deoxyribonucleotides in an TaqGOLD-supplied buffer.The samples were heated at 95° C. for 10 minutes to activate the enzyme,then cycled at appropriate temperatures and for the appropriate numberof cycles to achieve amplification of the designated target sequences,while remaining in the exponential phase of the reaction. For example,the samples are amplified for between 30-45 cycles using the followingtemperatures and times, 94° C. for 30 sec., 55° C. for 30 sec., and 68°C. for 1 min. See Innis, supra.

Example 6 Detection of Amplification Products by Mass Spectrometry

[0211] After PCR amplification, samples were ready for separation andanalysis. The method of ionization used for mass spectrometric analysiswas Matrix-Assisted Laser Desorption and Ionization (MALDI). Massdeterminations were made by Time-of-Flight (TOF). Adesorption/ionization matrix for analyzing samples was composed of a 9:1ratio of saturated hydroxypicolinic acid (HPA) to picolinic acid (PA)(Aldrich) in 25% acetonitrile and 25 mM diammonium citrate. A massspectrometer analysis plate was spotted in 384 positions with aliquotsof the matrix, which were then allowed to dry and/or crystallize. Adefined quantity of an oligonucleotide (e.g., 0.5 μl of a 5-10 AMsolution, depending on the mass of the oligonucleotide), having a masswithin the range of the amplification products, was added to each PCRreaction to serve as an internal quantitation standard. An aliquot ofapproximately 0.5-1 μl of each sample was then pipetted on top of eachof the crystallized spots. Samples were allowed to dry again, formingDNA:HPA co-crystals.

[0212] The sample plate was placed in the mass spectrometer load lockchamber, pumped down to a low vacuum pressure, transferred to the samplechamber, then finally pumped down further to the required operatingvacuum pressure. The sample chamber contains an X-Y table to orient thesamples under the laser beam, and ion optics to accelerate and directDNA ions into the flight tube and towards the detector. Ionized DNAfragments hitting the detector are assigned a mass based on the timerequired to travel through the flight tube. Various parameters were setwithin the automated data collection software to enable collection ofsignal in the appropriate mass range, and the coordinate positions onthe analysis plate for the samples to be examined were entered. A laserbeam of 355 nm light was focused through a window in the sample chamberonto the sample being analyzed. The laser power was adjusted to maximizethe signal-to-noise ratio, while minimizing fragmentation of DNA in thesample. Data was collected according to the set parameters, generating asignal spectrum for each sample. The data was further processed usingsignal calling software proprietary to GeneTrace. The software smoothedthe spectra, identified signal peaks, assigned masses to the peaks, andintegrated the data to quantitate the relative amount of each species inthe sample. These values were then normalized to the internalquantitation standard to convert the data to absolute values.

[0213] Data generated by the signal calling software was imported intoMicrosoft Excel (Bellevue, Wash.). Signals from each of the geneproducts being quantitated were normalized to the signal from thereference nucleic acid (the multiplex control target taken to have aconstant abundance level). When a second reference target was includedin the multiplex, this signal was also normalized to the firstreference, and checked to confirm that its abundance relative to thefirst reference was constant. Data was stored in tabular form asnormalized signal intensities.

[0214] Additional details regarding analysis by mass spectroscopy arepresented in further examples as detailed below.

Example 7 Multiplex Amplification Using a Single, Labeled ForwardUniversal Primer and an Unlabeled Reverse Universal Primer

[0215] The cDNA products of another toxicology multiplex sample wereused as templates in a PCR amplification that generated labeledproducts. A mixture of forward target-specific primers appropriate forthe multiplex reaction was prepared. These primers contained a differentuniversal sequence within their 5′ regions as that of the reverseprimers used to generate the cDNA. A forward universal primer wasmodified by covalent attachment of a fluorescein moiety (FAM, availablefrom Perkin-Elmer/Applied Biosystems, Inc.), while the reverse universalprimer remained unlabeled. The forward target-specific primers and theuniversal primers were diluted to a working concentration and then addedto samples from the reverse transcriptase reaction, along with TaqGOLDand deoxyribonucleotides in an appropriate buffer. The PCR amplificationwas carried out as described in Example 5.

Example 8 Generating a Pool of Two Multiplexed Amplifications—Using aSingle Forward Universal Primer Containing One of Two Labels and anUnlabeled Reverse Universal Primer

[0216] The cDNA products of additional toxicology multiplex samples wereused as templates in two PCR amplifications to generate differentlylabeled products. A mixture of forward target-specific primersappropriate for the multiplex reaction was prepared. These primerscontained a different universal sequence within their 5′ regions as thatof the reverse primers used to generate the cDNA. In addition to thefluorescein-modified primer described in Example 2, a second preparationof the forward universal primer was made, modifying it by covalentattachment of a hexachlorofluorescein moiety (HEX, Perkin-Elmer/AppliedBiosystems, Inc.). The reverse universal primer remained unlabeled. Theforward target-specific primers and the universal primers were dilutedto a working concentration (of 10 nM and 500 nM respectively). Forwardtarget-specific primers, TaqGOLD and deoxyribonucleotides in anappropriate buffer were added to samples from the reverse transcriptasereaction. The FAM-modified forward universal primer was added to one ofthe PCR amplification reactions, and the HEX-modified forward universalprimer was added to the other. PCR amplification was carried out asdescribed in Example 5.

Example 9 Detection of Amplification Products by Polyacrylamide GelElectrophoresis

[0217] After PCR amplification using the fluorescently-labeled primers,the multiplexed samples were ready for analysis by polyacrylamide gelelectrophoresis. A standard sequencing gel composed of 5%polyacrylamide, and containing 6M urea and 890 mM Tris-borate and 2 mMEDTA, was cast for use on an ABI PRISM 377 DNA Sequencer(Perkin-Elmer/Applied Biosystems). Amplification products were dilutedand mixed with a solution of GeneScan 500 ROX-labeled size standards (PEApplied Biosystems, CA) in formamide (1:5). Samples were loaded on thegel, and the components of the multiplex reaction mixture wereelectrophoretically separated by size according to standard conditions,for example, 1.5 hours running at 2000 V, 60 mA current, 20 W power, geltemperature of 51° C., and laser power of 40 mW (ABI 377). Fluorescentdata was collected by laser scanning across the gel in real time.GeneScan™ software was used to quantitate fluorescent signals from theamplification products, and Genotyper™ software (both fromPerkin-Elmer/Applied Biosystems) was used for subsequent calculationsand data manipulations.

Example 10 Generating a Pool of Two Multiplexed Amplifications—Using TwoForward Universal Primers of Different Lengths and With DifferentLabels, and an Unlabeled Reverse Universal Primer

[0218] The cDNA products of other toxicology multiplex samples were usedas template in two PCR amplifications to generate equivalentamplification products of slightly offset sizes, both labeled with thesame chromaphore. A mixture of forward target-specific primersappropriate for the multiplex was prepared. These primers contained adifferent universal sequence at their 5′ ends as that of the reverseprimers used to generate the cDNA. Two forward universal primers weremade with the same universal sequence, but one contained threeadditional bases at its 5′ end. One of the forward universal primers wasmodified by covalent attachment of a FAM moiety, and the other wasmodified by covalent attachment of a HEX moiety. The reverse universalprimer remained unlabeled. The forward target-specific primers and theuniversal primers were diluted to a working concentration. Forwardtarget-specific primers, TaqGOLD and deoxyribonucleotides in anappropriate buffer were added to samples from the reverse transcriptasereaction. One of the labeled forward universal primers was added to eachof the reactions. PCR amplification was carried out as described inExample 4.

Example 11 Detection of Amplification Products by Denaturing CapillaryElectrophoresis

[0219] Two PCR multiplex samples are analyzed by capillaryelectrophoresis at the end of the PCR amplification. The samples werecombined, diluted 1:10 in CE sample dilution buffer (1:5 dilution offluorescently labeled ladder in deionized formamide). The pooled samplewas analyzed on an ABI PRISM 310 Genetic Analyzer, with capillariescontaining POP4 acrylamide matrix (PE Perkin-Elmer Applied Biosystems,CA). Components of the pooled multiplexes were electrophoreticallyseparated by size according to standard conditions. Fluorescent data wascollected at wavelengths appropriate for the FAM and HEX labels. Sizeswere assigned to each signal peak based on their migration relative tothe ROX size standards.

Example 12 Data Analysis

[0220] The data collected from the FAM and HEX fluorescent signals wereanalyzed using GeneScan analysis software. The fluorescent signals weredeconvoluted to yield information specific for each of the individualfluorophores in the mixture, to generate a baseline, to sort the signalsinto “size bins” relative to the ROX size standards, and to quantitatethe amount of DNA represented in each bin. The results from thisanalysis were further processed by Genotyper software (PE AppliedBiosystems, CA) to automate the repetitive tasks of data analysis.Sample files from GeneScan were imported into Genotyper, which thenassigned data to the size ranges programmed by the operator. The datagenerated in this manner was stored in tabular form, and then importedinto Excel. The signals from each of the gene products being quantitatedwere normalized to the signal generated by the internal reference (themultiplex control target taken to have a constant abundance level). Whena second internal reference target was included in the multiplex, thissignal was also normalized to the first reference, and checked toconfirm that its abundance relative to the first reference was constant.Data was stored in tabular form as normalized signal intensities.

Example 13 Multiplex Analysis of Cellular Transcription in PC-3 CellsAfter Treatment With Battery of Compounds

[0221] Preparation of Target Sequences

[0222] PC-3, a human prostate adenocarcinoma cell line (American TypeCulture Collection, Rockville, Md.) was cultured in T-225 cm² flasks(Corning Costar Corp., Cambridge, Mass.) using Kaighn's Nutrient MixtureF-12 (Irvine Scientific, Santa Ana, Calif.) containing 7% fetal bovineserum (FBS) (Hyclone, Logan, Utah) and 1 mM L-glutamine. The cellculture reagents were obtained from Gibco BRL Life Technologies (GrandIsland, N.Y.) except where otherwise noted. Cells were maintained at 37°C. in a humidified cell incubator containing 5% CO₂. At approximately70% confluence, the growth media was aspirated and cells were rinsedwith D-PBS. Cells were harvested by trypsinization, treated with trypanblue exclusion viability stain and counted using a hemacytometer. Lidded96-well microtiter culture plates (Becton Dickinson, Franklin Lakes,N.J.) were then seeded at 5×10⁴ cells per well in a 200 μL media volume.Two wells were left empty to allow the later addition of externalprocess controls. Seeded plates were incubated for 3 hours (37° C., 5%CO₂, in a humidified cell incubator) to allow for cell attachment priorto compound addition.

[0223] A set of 80 known drugs (“Killer Plate 1”, from MicroSourceDiscovery Systems, Inc., Gaylordsville, Conn.) and an actinomycin-Dpositive control were solubilized in 100% DMSO (Sigma Chemical Co., St.Louis, Mo.) and diluted to 8×working solutions with growth media priorto cell plate addition. Compounds from a chemical library (in pooledformat) and subsequent confirmation of individual compound activitieswere analyzed at a final concentration of 2.5 μM in 0.25% DMSO. Positiveand vehicle control wells were maintained at 0.25% DMSO (v/v) which hadno effect on cell growth or gene targets. For dose-response analysis,compounds were plated in triplicate and analyzed using eightconcentrations (between 10 μM and 3.16 nM in 0.25% DMSO), as prepared byserial dilution. After cell attachment was verified by phase contrastmicroscopy, a 25 μL aliquot of media was removed from the cell plate andan equivalent volume of compound working solution (8×) was introducedwith mild trituration of the well volume, using a MultiMek 96 pipettingstation (Beckman Coulter, Fullerton, Calif.). Cell plates were thenreturned to the incubator for a 24 hour exposure period.

[0224] Lysis buffer was prepared by adding 145 mM β-mercaptoethanol(Sigma Chemical Co., St. Louis, Mo.) and external mRNA controls (to afinal concentration of 500 fM) to RLT Lysis buffer (Qiagen, Valencia,Calif.). Two external mRNA controls were used: 7.5 kb poly(A)-tailed RNAand 1.2 kb Kanamycin Positive Control, which were treated with DNAse toensure that no contaminating DNA was present. Following a 24 hourincubation period, cell media was aspirated from all wells using anEL-404 plate washer (BioTek Instruments, Winooski, Vt.). Lysis buffer(100 μL) was pipetted into each well containing cells. Plates were thenmixed on an orbital shaker (Labline, Melrose Park, Ill.) for 15 seconds.Adhesive aluminum foil strips (E&K Scientific, Campbell, Calif.) wereused to seal the plates prior to frozen storage at −20° C.

[0225] For gene expression analysis, the cell lysates were thawed, andtotal RNA was purified in automated 96-well format using the QiagenRNeasy 96 kit according to the manufacturer's recommended procedure. RNAconcentrations were determined fluorometrically using RiboGreen reagent(Molecular Probes, Eugene, Oreg.), adjusted in concentration, andaliquoted in 30 ng amounts into 96-well plates for assay. Total RNAyields ranged from 0.45 to 1.8 μg per well depending on compoundtoxicity. RNA samples were verified to be free from DNA contamination byrunning controls in which MMLV reverse transcriptase enzyme was omittedfrom the multiplex assay protocol. Purified RNA controls were includedon each plate for process quality control and tracking.

[0226] Primer Design

[0227] Assay specificity was determined by utilizing unique primers foreach gene. Target-specific primers were designed to six target sequencesand two reference sequences (Table 1). Both forward-TSPs and reverseTSPs were synthesized, having sequences as delineated in Table 2. The 5′region of the target-specific sequences includes sequences complementaryto one of two universal sequences TABLE 1 Target-Specific Primers forMultiplexed Analysis of Gene Expression in PC-3 cells Target SequenceF-primer R-primer Size (bp) beta-actin Sp61F T7(P7)R3/R3pi (1:39) 117cloning vector lambda EMBL3 SP6/T7 fragment in GibcoBRL 7.5 kp mRNASp6(P2)F2 T7(P7)R2 127 INA D Sp6F1 (P2) T7R1 (P7) 147 hSPE Sp6F2 (P2)T7R2 (P7) 157 Sp6F1 (&F2) survivin (P2) T7R2 (P7) 200 HNF 3 alpha Sp6F3(P2) T7R3 (P7) 215 GAPDH Sp6F1 (P2) T7R1 (P7) 237 EST Sp6(P2)F4 T7(P7)R4266 Hoxb 13 Sp6F1 (P2) T7RL(&R2) (P7) 283 (KanR) aminoglycoside 3′-Sp6(P2)(LP70)F2 phosphotransferase T7(P7)R2 322

[0228] TABLE 2 Target-Specific Primer Sequences Accession # PrimerPrimer Name Primer Sequence X00351 β-actin forward Sp6.1F1AGGTGACACTATAGAATAACCGAT AAGGCCAACCGCGAGAAGATGA X00351 β-actin reverseT77R3 GTACGACTCACTATAGGGATGGAT β-actin reverse AGCAACGTACATGGCTG X00351Phosphorylated T77R3Pi GTACGACTCACTATAGGGATGGAT U02426AGCAACGTACATGGCTGPi fragment 7.5 kb forward Sp6 (P2) F2AGGTGACACTATAGAATAACTATG U02426 CCGGTATCAGCACC fragment 7.5 kb reverseT7 (P7) R2 GTACGACTCACTATAGGGAGATGG CAGCGTGATTTCAC INA D INA D forwardSp6F1 (P2) AGGTGACACTATAGAATAGTGACA CGTCGCAGAATGAG INA D INA D reverseT7R1 (P7) GTACGACTCACTATAGGGATTGAC CCTTCAGTTGCTTGA hSPE hSPE forwardSp6F2 (P2) AGGTGACACTATAGAATAGCTTCA TTAGGTGGCTCAACA hSPE hSPE reverseT7R2 (P7) GTACGACTCACTATAGGGAGGCTC Survivin Sp6F1 (& F2) AGCTTGTCGTAGTTCSurvivin forward (P2) AGGTGACACTATAGAATAGTCAGC CCAACCTTCACATC SurvivinSurvivin reverse T7R2 (P7) GTACGACTCACTATAGGGACCACC HNF 3 alphaCTGCAGCTCTATGAC HNF 3 alpha forward Sp6F3 (P2) AGGTGACACTATAGAATAACTTCAHNF 3 alpha AGGCATACGAACAG HNF 3 alpha reverse T7R3 (P7)GTACGACTCACTATAGGGAGGGAG GAPDH CTAGGAAGTGTTTAG M33197 forward Sp6F1 (P2)AGGTGACACTATAGAATAAAGGTG AAGGTCGGAGTCAA M33197 GAPDH reverse T7R1 (P7)GTACGACTCACTATAGGGAATGAC GAPDH reverse AAGCTTCCCGTTCTC M33197phosphorylated T7R1Pi (P7) GTACGACTCACTATAGGGAATGAC AAGCTTCCCGTTCTCPiEST EST forward Sp6F4 (P2) AGGTGACACTATAGAATAGCTCAT CTGCCAACAATC EST ESTreverse T7R4 (P7) GTACGACTCACTATAGGGACTAGC Hoxb 13 GGAAGCAAATTACAC Hoxb13 forward Sp6F1 (P2) AGGTGACACTATAGAATAGCGACA T7R1 (&R2) TGACTCCCTGTTHoxb 13 Hoxb 13 reverse (P7) GTACGACTCACTATAGGGAAACTT J01839 Sp6 (P2)(LP70) GTTAGCCGCATACTC (V00359) KanR forward F2 AGGTGACACTATAGAATAATCATCJ01839 AGCATTGCATTCGATTCCTGTTTG (V00359) KanR reverse T7 (P7) R2TACGACTCACTATAGGGAATTCCG ACTCGTCCAACATC

[0229] Preparation of Primer Sequences

[0230] Oligonucleotides were prepared using phosphoramidite methodologyon an ABI 394 DNA synthesizer using standard procedures and reagents,including dG^(dmf) FastPhosphoramidite (PE Biosystems 401183), 0.02MIodine (PE Biosystems 401732) as oxidant, and 0.25M 5-ethyl-1H-tetrazole(Glen Research 30-3140-52) as activator. 5′-biotinylated nucleotideswere incorporated using commercially available amidite reagents asdescribed in the procedure below. Preparation of the cleavable primersequences involved the synthesis of a protected 3′ thiothymidine reagent(5′-O-Dimethoxytrityl-3′-thiothymidine-3′-S-(2-cyanoethyl)-N,N-diisopropylphosphorothioamidite). The 3′-thiothymidine nucleotide was incorporatedin an automated fashion using the protected phosphoramidite reagentdescribed above. Column chromatography was carried out under a positivepressure of argon gas. HPLC data were collected on an Hewlett-Packard1100 series instrument at 260 nm.

[0231] In cases where mass spectrometric analysis was performed, oneuniversal primer of each target-specific primer pair was prepared havinga biotin moiety incorporated at the 5′-end, and a chemically-cleavablebase, 3′-thiothymidine at an appropriate position. Cleavage of theamplified PCR product at the position of the 3′-thiothymidine reducesthe measured DNA size, thus providing fragments suitable for optimalmass spectral resolution and sensitivity. Furthermore, the cleavablebases could be introduced in various positions within differentuniversal primers used in different multiplex reactions. The variouscleaved positions yield a series of non-overlapping mass spectral peakssuitable for multiplexed readout.

[0232] 5′-biotin phosphoramidite (Glen Research 10-5950-90, 0.1M inanhydrous acetonitrile) and Thio-T amidite (0.1M in anhydrousacetonitrile) were employed in the synthesis of the universal primers.The synthesis was carried out using a 10-minute coupling time for Biotinand a two 5-minute couplings for Thio-T. The crude oligonucleotide wasdeprotected in 28% aqueous NH3 at 55° C. for two hours. Removal of thesolvent gave a white residue that was desalted on a NAP-10 column(Pharmacia 17-0854-01) with ddH2O. The product was analyzed by HPLCusing a Supelcosil LC-18-T column (Supelco 58971) and a gradient of 10to 20% acetonitrile from 5 to 25 min. at 1 mL per min. in 0.1M TEAA.Typical retention times were about 10 to 15 min., and the purity of theproduct should exceed 80%.

[0233] For cases where the samples were analyzed on a fluorescenceelectrophoretic device, a universal primer was synthesized that includeda dye at the 5′ end. Fluorescent dye labeling of primers with 6-FAM wascarried out on an automated DNA synthesis device using 5′-fluoresceinphosphoramidite (Glen Research, Sterling, Va.). “Shifted” UniversalPrimers

[0234] Greater assay throughput is achieved by mixing PCR products ofthe original gene set (i.e. target sequences) with a “shifted” gene setso that signals from the products of the two gene sets are interleaved.The “shifted” genes are separated from the original genes by the samenumber of bases for each product in the multiplexed gene set. The“shifted” genesets are generated by the addition of nucleotides to thelabeled strand of the universal primer to increase the length of the PCRproducts. Spacers are used to separate the label from the specificportion of the universal primer sequence.

[0235] Shifted target universal primers were synthesized that containeda normucleotide linker. The normucleotide linker used was an abasicnucleotide, dSpacer phosphoramidite,5;-dimethoxytrityl-1,2-dideoxyribose-3′-cyanoethyl phosphoramidite (GlenResearch, Sterling, Va.) The dSpacer was incorporated during automatedDNA synthesis on a DNA synthesis device using standard methods. Afterincorporation of the dSpacer between 1 to 10 thymidine bases wereincorporated and optionally a dye label was also added.

[0236] For example, the universal primers used in a first series ofmultiplex amplifications to generate an original geneset comprises aFAM-labeled Sp6 universal sequence (forward direction) and an unlabeledT7 universal sequence (reverse direction)

[0237] Labeled Sp6: 5′-(FAM)-AGG TGA CAC TAT AGA ATA-3′ (SEQ ID No. 23)

[0238] Non-labeled T7: 5′-GTA CGA CTC ACT ATA GGG A-3′(SEQ ID No. 24)

[0239] Alternatively, the T7 sequence can carry the fluorescent labelwhile the Sp6 sequence is unlabelled: Non-labeled Sp6: 5′-AGG TGA CACTAT AGA ATA-3′ Labeled T7: 5′-(FAM)-GTA CGA CTC ACT ATA GGG A-3′

[0240] In a second set of multiplex amplifications, universal primerscontaining additional nucleotides are employed (dS=dSpacerphosphoramidite, available from Glen Research, Sterling Va.), such thatthe molecular weight or mass of the resulting amplified sequences isaltered as compared to the first series of amplification reactions.Exemplary universal primers for generation of the shifted geneset are:Labeled Sp6: 5′-(FAM)TTTTTTT-dS*-AGG TGA CAC TAT AGA ATA-3′ Non-labeledT7: 5′-GTA CGA CTC ACT ATA GGG A-3′

[0241] As with the primers used in the previously-describedamplification reaction, the label can be carried on either of theuniversal sequences employed:

[0242] Non-labeled Sp6: 5′-AGG TGA CAC TAT AGA ATA-3′ Labeled T7:5′-(FAM)-TTTTTTT-dS*-GTA CGA CTC ACT ATA GGG A-3′

[0243] Reactions may also be performed separately for the same set oftarget sequences using multiple dyes, which are then mixed to increasethroughput. Labeled universal primers are also “shifted” in size toavoid overlapping peaks and for improved reproducibility. All reactionsusing multiple dyes were performed with the same non-labeled T7universal primers. Exemplary labeled Sp6 universal primers include:FAM-labeled Sp6: 5′-(FAM)-AGG TGA CAC TAT AGA ATA-3′ HEX-labeled Sp6 :5′-(HEX)-TAG AGG TGA CAC TAT AGA ATA-3′ or 5′-(HEX)-TTT-(dS)-AGG TGA CACTAT AGA ATA-3′ NED-labeled Sp6: 5′-(NED)-GAT TAG AGG TGA CAC TAT AGAATA-3′

[0244] Additional primers can be designed by one of skill in the art.For example, reactions may also be performed where one of the universalprimers contains a cleavable site and optionally a biotin, for specificsolid-phase capture. Cleavable universal primers are “shifted” in sizeonce they are cleaved. As an example, all reactions using cleavable Sp6primers were performed with a non-labeled T7 universal primer. Exemplarylabeled Sp6 universal primers include: Cleavable Sp6: 5′-(Biotin)-AGGTGA CAC TAthioT AGA ATA-3′

[0245] Amplification

[0246] The multiplex amplification step utilized solution-phasequantitative multiplex RT-PCR amplification, and was coupled withmultiplexed fluorescence or mass spectrometric detection. Primer pairs(SEQ ID Nos. 1-22) for specific genes and controls were designed usingPrimer-3 software (Whitehead Institute for Biomedical Research,Cambridge, Mass.).

[0247] Reverse transcription to generate first strand cDNA was carriedout using 30 ng of total RNA, 0.02 μM primers, 1 mM dNTPs, RNasinribonuclease inhibitor (2.5 units, Promega, Madison, Wis.), and MMLVreverse transcriptase (10 units, Promega, Madison, Wis.) at 42° C. for30 minutes. PCR amplifications were performed using 0.01 μMgene-specific primers, 1 μM universal primers, 0.375 mM dNTPs (Promega,Madison, Wis.), and AmpliTaq Gold polymerase (1 unit, Perkin Elmer,Foster City, Calif.) in the buffer supplied with the enzyme. Thermalcycling was performed on a Perkin-Elmer GeneAmp 9700 between 30 to 45cycles using the following conditions: 94° C. for 30s, 55° C. for 30s,and 68° C. for 1 minute. Multiplex PCR products were resolved usingeither the electrophoresis or capillary systems for fluorescent readoutwhen they were all in the linear range of amplification, and werequantified by fluorescence intensity. For fluorescent readout, one ofthe universal primer pairs used for PCR amplification was labeled withthe fluorescent dye 6-FAM utilizing 5′-fluorescein phosphoramidite (GlenResearch, Sterling, Va.).

[0248] Gel electrophoresis

[0249] The samples were prepared for multiplex fluorescent readout usinga gel electrophoresis system from The Gel Company (San Francisco,Calif.) by diluting the RT-PCR products 1:4 in GE sample dilution buffer(a 1:3.3 dilution of fluorescently labeled ladder (CXR FluorescentLadder, Promega, Madison, Wis.), 1:16 dilution of blue dextran, and1:1.6 dilution of deionized formamide). The fluorescent ladder was usedas a gel standard with every sample for normalization of the target PCRproduct sizes. After denaturing the samples at 95° C. for 5 minutes andcooling in ice-water bath for 5 minutes, 0.5 μl of the diluted RT-PCRsamples were loaded onto a 96-well linear loading tray and transferredvia absorption onto a 96-lane paper comb. The comb was then insertedonto the gel and samples were allowed to run into the gel forapproximately 35 seconds, after which the comb is removed and discarded.

[0250] Capillary electrophoresis

[0251] RT-PCR products for multiplex fluorescent readout using thecapillary electrophoresis system were diluted 1:10 in CE sample dilutionbuffer (1:5 dilution of fluorescently labeled ladder in deionizedformamide). Approximately 10 μl of the diluted RT-PCR samples wereplaced in receptacles specific for the capillary electrophoresisinstrument and denatured at 95° C. for 5 minutes. The samples were thencooled for 5 minutes in ice-water bath prior to performing the capillaryelectrophoresis.

[0252] Mass Spectroscopic Analysis

[0253] Subsequent to PCR amplification, samples were processed toprepare them for mass spectrometric analysis. The processing steps wereconducted in 384-well plates on a robotic workdeck containing a magneticplatform to facilitate manipulation and washing of magnetic beads.

[0254] Streptavidin-coated magnetic beads were added to each sample inbinding solution, 10 mM Tris, 20 mM ammonium acetate, 1 mM EDTA buffer,pH 7.2, and incubated at room temperature for 20 minutes to allowbinding of the biotinylated primer. The sample tray was placed on amagnet platform of a robotic workstation to precipitate the DNA bound tothe beads. After the beads were pelleted, the supernatant was removed,and the pellet was rinsed once with binding solution.

[0255] A denaturing solution of 0.1N NaOH was used to rinse the pelletedbeads and to remove the non-biotinylated complementary strand. A secondaliquot of the denaturing added, mixed above the pelleted beads, thenincubated. The mixing process was repeated four times, then the finalsupernatant was removed. The beads were washed five times with a 20 mMammonium acetate solution, then twice with deionized water to removeresidual salts. The beads were then resuspended in a cleavage solution(0.1 mM silver nitrate) land the samples were incubated at 48° C. for 15minutes. The tray was returned to the workstation to precipitate thebeads, and the supernatant was transferred to a fresh 384-well tray. Asolution of 70 mM DTT solution was added to samples in the new tray toquench the reaction, and samples were dried in a vacuum centrifuge.

[0256] Approximately 0.5 mL of a matrix solution consisting of a 5:1molar ratio of 3-hydroxypicolinic acid (3-HPA) to picolinic acid (PA)was added to each well containing dried sample. The matrix solution wasprepared by mixing 18 μL of a freshly prepared saturated 3-HPA solution(about. 0.5 M) with 2 μL of 1 M PA. The redissolved samples were thenspotted (either manually or robotically) onto a mass spectrometer sampleplate, 0.5 μl, and allowed to crystallize for subsequent analysis.

[0257] For mass spectrometry readout, a linear time-of-flight (TOF) massspectrometer was employed, using an acceleration voltage of +20 kV;delay of +3.6 kV at 1.12 μsec; laser setting of 179 on the polarizer;mass gate of 5.84 μsec; and 400 shots. Furthermore, a 2-point masscalibration with a 15-mer (4507.0 Da) and a 36-mer (10998.2 Da) wasutilized.

[0258] Quantitative levels of all genes in each sample, including targetand external spike control genes, were normalized to the internalcontrols, and are expressed as ratios to the control (“housekeeping”)genes GAPDH and β-actin.

[0259] Validation of Primer Design

[0260] Multiplexed amplifications were validated to ensure that eachprimer pair was specific for a particular target sequence and that therewere no interactions among the target sequences in the multiplex. Thiswas accomplished by conducting drop-out experiments, in which themultiplex amplification was run in the absence of a particular primerpair. Additionally, the amplification reaction was validated bycomparing the results of primers in different multiplex environments,ensuring identical PCR product sizes in each case. Furthermore, primerswere also tested for efficiency by running the multiplex assay on RNAsamples known to express all of the targeted sequences.

Example 14 Multiplex Strategies

[0261] Table 3 depicts exemplary strategies for multiplexing samples inthe methods of the present invention. Multiplex reactions A and Billustrate fundamental multiplexing strategies for use in the methods ofthe present invention. In these assays, all of the forward universalprimers (UPfs) include the same universal sequence; in addition, asingle type of dye label is incorporated into the primers. In multiplexreaction A, the reverse universal primers (UPrs) all have the samesequence with each other, but a different sequence from the forwarduniversal primers. The reverse universal primers do not have anincorporated dye. In multiplex reaction B, all of the forward universalprimers and reverse universal primers contain the same sequence, andtherefore both strands of the products will have an incorporated dye. Inthe given example, at the end of each type of reaction, the multiplexedsamples contain 12 strands of amplified products (two complementarystrands from each of six templates), with dye incorporated in eitherhalf (for example A) or all (multiplex reaction B) of the strands.Because the dye is the same for all targets, detection of individualproducts depends on their separation (in this case, based on size).

[0262] Multiplex reaction C depicts an embodiment in whichsemi-universal primers are used to shift the mobility of a subset of theamplification products during size separation of otherwise overlappingpeaks. Two forward universal primers are used for designated subsets oftargets. Both primers are labeled with the same dye, but one of themadditionally contains a friction group (i.e., an attached moiety thatgenerates drag on molecules as they migrate through a non-matrixed,liquid solution). See, for example, Hubert and Slater (1995)Electrophoresis 16:2137-2142. In this example, the sizes of products 1and 4, 2 and 5, and 3 and 6 are the same or overlapping, butcorresponding peaks 1, 2, and 3, as well as 4, 5, and 6 are differentsizes. The friction group will be incorporated into products 4, 5, and6, while leaving products 1, 2, and 3 unmodified. As a result, themobilities of products 4-6 will be retarded relative to 1-3, resolvingthese otherwise overlapping sets into six separate peaks. Theillustration represents the reverse universal primers as all being thesame sequence, but these primers may also comprise a set ofsemi-universal primers.

[0263] Multiplex reaction D illustrates another embodiment of thecomponents of the multiplex reaction which can be employed in order toresolve overlapping signals. In this reaction profile, two amplificationproducts of the multiplex are the same size. The mobility of one of thetwo overlapping signals can be shifted by adding a nucleic acid sequenceto one or both of the TSPs for one of the target sequences, lengtheningits amplification product. A similar effect is obtained by designingsemi-universal primers of different sizes.

[0264] Multiplex reaction E illustrates an important embodiment of themethods of the present invention, which provides a mechanism by whichthe signals for multiple species are resolved by separating other thanby size. A set of semi-universal primers is employed in the multiplexreaction; each UPf is labeled with one of a set of independent labels,each of which can be detected uniquely. As with multiplex reaction C,the sizes of products 1 and 4, 2 and 5, and 3 and 6 are taken as thesame or overlapping, but peaks 1, 2, and 3, as well as 4, 5, and 6 aredifferent sizes. Products 1-3 will be labeled with dye number 1, andproducts 4-6 with dye number 2. The two sets of three products willstill have overlapping mobilities, but the fluorescent signals given byeach of the two dyes can now be separated by deconvolution of theemission spectral data. As in the previous example, the UPrs can also bedesigned as semi-universal primers.

[0265] Multiplex reaction F illustrates a method for obtaining signalsfrom a greater number of unresolved species than the number of availabledyes. Two dyes were used in the multiplex illustration of multiplexreaction E, enabling resolution of two overlapping signals. In theembodiment described in multiplex reaction E, the signal from threeunresolved products are obtained using only two dyes with threedifferent UPf primers. In this embodiment, the third signal is obtainedby double-labeling the amplification products of that target. Becausethe signal from this product is known to contain an equivalentfluorescent signal from each of the two dyes, its signal can beseparated from the signals of the two singly-labeled products. Thisapplication requires that the three types of products are not completelyoverlapping, which would make deconvolution of their signals verydifficult. Ideally, the signals from the two singly-labeled speciesshould not overlap, but some overlap can be resolved by signalprocessing of the data. More complex combinations are obviously possiblewhen more than two dyes are used.

[0266] These six cases are provided for illustration of the moreimportant embodiments of multiplexing reactions described in thisinvention. To one skilled in the art, many variations in multiplexingstrategies are possible by combining separate elements of theseexamples. In particular, combination strategies can be employed makinguse of the separate forward and reverse universal primers, or thecombinations of target-specific and universal primers, or semi-universalprimers. In all cases, the selection of the particular TSP sequences foreach target within a multiplex can be performed carefully to select thesize of each PCR product and ensure that each product can be detecteduniquely.

[0267] Optionally, the methods of the present invention include methodsto increase the number of samples simultaneously analyzed by pooling theproducts of separate reactions. This strategy increases the throughputand reduces the cost of the assay for situations in which the pooledproducts cannot be generated in the same reaction (for example, wheneach separate reaction is already maximized in multiplexing potential).For example, samples are pooled after the RT-PCR reaction is complete,and prior to analysis and quantitation. TABLE 3 Multiplexing Strategiesfor the RT-PCR Example UPf UPf label UPr UPr label Application A UPf 1-6= dye #1 UPr 1-6 = none Resolution of a simple multiplex sequence “A”sequence “B” by size (two universal primers) B UPf 1-6 = dye #1 UPr 1-6= dye #1 Resolution of a simple multiplex sequence “A” sequence “A” bysize (one universal primer) C UPf 1-3 set, dye #1 UPr 1-6 = none Usesemi-universal primers to sequence “A” sequence “B” create resolution byaffecting + mobility UPf 4-6 set, dye #1 + Create resolution by sizeshifting sequence “A” friction (where amplification products 1-3 grouphave overlapping masses with products 4-6) D UPf 1-6 = dye #1 UPr 1-6 =none Create resolution by shifting size sequence “A” sequence “B” (TSPlength was changed to shift the mass of it's amplicon) E UPf 1-3 = dye#1 UPr 1-6 = none Use semi-universal primers to sequence “A” sequence“C” resolve by size & fluorescence UPf 4-6 set, dye #2 (multiplexingwith dyes) sequence “B” F UPf 1 = dye #1 UPr 1-6 = none Increase dyemultiplexing capacity sequence “A” sequence “D” possible with a fixednumber of + dyes UPf2 = dyes #1 sequence “B” and 2 + (50:50) UPf3 = dye#2 sequence “C”

Example 15 Pooling of Samples Using Interleaving Genesets or MultipleDyes

[0268] RT-PCR samples for the same multiplexed reaction may be mixed atappropriate ratios by combining either the original set of targetsequences with the “shifted” target sequence set, and/or by combiningreactions with multiple dyes. These mixed samples are then diluted inthe appropriate sample dilution buffer and loaded onto the gel orcapillary electrophoresis system. Exemplary profiles of original and“shifted” multiplex genesets are shown in FIG. 4. Examples of profilesgenerated by multiplexed amplification with different dyes usingmultiplex genesets are shown in FIG. 5. Several illustrations of poolingstrategies are listed in Table 4, and described below.

[0269] Multiplex reaction G illustrates an embodiment of a fundamentalpooling strategy for use in the methods of the present invention. Inthis example, two separate reactions (G1 and G2) comprise differentmultiplexes. The combined products of the two separate reactions areresolvable by size. (For examples G through M, it is assumed forillustration that all of the products of each separate multiplex areresolvable by size.) As an example, each separate reaction may beperformed with the same UPf primer, labeled with the same chromaphore.After the reaction, the samples are combined for analysis. All of theindividual signals from the two reactions are then resolved by size.

[0270] The embodiments provided in Cases H-L illustrate various ways ofresolving the same set of amplified sequences generated in separatereactions. Multiplex reaction H illustrates the use of isotopic orchemical modification to generate shifts in the masses of otherwiseequivalent amplification products. For example, deuterated dNTPs may beused to generate “heavy” amplification products (designated as sequenceA^(H) in reaction H2) in one reaction, while unmodified dNTPs are usedin another (reaction H1). The heavier deuterium isotopes of hydrogenthat are incorporated in one set of reaction products will generate ashift in the mass of each product relative to the equivalent amplicon ofthe other reaction.

[0271] The embodiment illustrated with multiplex reaction I makes use ofthe friction molecules described previously in multiplex reaction C. Inmultiplex reaction I, two reactions (I1 and I2) of the same multiplexset are performed, the first with unmodified UPf primers and the secondwith UPf primers containing a friction group. Both primers are labeledwith the same dye. After the reaction, samples are combined foranalysis. The friction group will be incorporated into all of theproducts of reaction I2. As a result, the otherwise overlapping signalswill be separated by the frictional drag of one species relative to theother.

[0272] Multiplex reaction J provides a way for detecting duplicatemultiplex sets by a mass shift. In this embodiment, two UPf primers areused, one of which is shorter (in reaction J1) than the other (reactionJ2). Two separate reactions are conducted, each using differentuniversal primers. This will result in a duplicate signal pattern inwhich one group is offset from the other by a fixed size. This sizeoffset can also be accomplished by using two UPf primers coupled withtwo UPr primers, and changing the lengths of one pair of UPf and UPrprimers by a lesser amount.

[0273]FIG. 4 depicts exemplary detection profiles of original and“shifted” multiplex genesets, as prepared by methods of the presentinvention. The position of the signal along the X-axis generallycorrelates with number of nucleotides in the amplified product, whilethe Y axis indicates intensity of fluorescent signal. Panel A representsdata as collected for an “original” geneset, while panel B depicts datafor a “shifted” geneset (for which, in this example, the amplifiedproducts appear to have a greater mass or friction coefficient ascompared to the unmodified amplification sequences). Panel C presentsthe original and shifted genesets together, demonstrating the resolutionintroduced into the products of the “shifted” amplification reaction.

[0274] Multiplex reaction K illustrates a pooling strategy based on amass shift between duplicate multiplex sets, just as with multiplexreaction J. In this illustration primers of the same sequence and lengthare used for both multiplexes. However, for one of the reactions (K2),the UPf incorporates a site of cleavage between two nucleotides in theextension product. (Thus, the label must be incorporated 3′ to thecleavage site in order for it to remain with the extension product).After amplification is complete, the products made with the modifiedprimer are cleaved, removing a fixed number of nucleotides from the 5′end of the labeled strand. Cleavage may be performed after pooling ofseparate reactions. Cleavage sites can be situated in one of severalpositions in a primer sequence, facilitating pooling of multiplereactions.

[0275] In the embodiment illustrated in multiplex reaction L, identicalmultiplexed reactions are generated (reactions L1, L2 and L3). Ratherthan mixing the reactions prior to loading on the separation platform,they are simply loaded individually, but with time delays, in order togenerate an offset in their relative positions in the separation medium.

[0276] Multiplex reaction M illustrates the use of multiple labels, e.g.fluorescent dyes, each of which can be uniquely detected. In thisembodiment, three separate reactions (M1, M2 and M3) are performed witha single UPf primer sequence, but that contains one of three differentlabels. After the reaction, the three samples are combined for analysis.Each particular target from each reaction will have the same size asthose from each of the other reactions. The triplicate sets of signalsfrom the three reactions will be resolved by deconvolution of thefluorescence data. Examples of profiles generated by multiplexedamplification with different dyes using multiplex genesets are shown inFIG. 5. The position of the signal along the X-axis correlates withnumber of nucleotides in the amplified product, while the Y axisindicates intensity of fluorescent signal. Panel A=FAM-labeled products;panel B=HEX-labeled products; panel C=NED-labeled products; and panelD=FAM, HEX, & NED-labeled products combined. As with all other caseillustrations, the UPr primers can be utilized in conjunction with theUPf primers to design more complex strategies. TABLE 4 PoolingStrategies for Analysis Reaction UPf (product) Label UPr (product) LabelApplication G1 UPf 1-6 (seq A) dye #1 UPr 1-6 (seq B) none Resolution bysize. G2 UPf 7-12 (seq B) dye #1 UPr 7-12 (seq B) none H1 UPf 1-6 (seqA) dye #1 UPr 1-6 (seq B) none Separate reactions have relative mobilityshifts from use of different H2 UPf 1-6 (seq A^(H)) dye #1 UPr 1-6 (seqB) none isotopes I1 UPf 1-6 (seq A) dye #1 UPr 1-6 (seq B) none Separatereactions have relative I2 UPf 1-6 (seq A) dye #1 + UPr 1-6 (seq B) nonemobility shifts resulting from the friction “friction” group. (Note:product masses group J1 TSP f, set #1 (seq A) dye #1 UPr 1-6 (seq B)none Separate reactions have relative mass off sets resulting fromprimer J2 TSP f, set #2 (seq A + dye #1 UPr 7-12 (seq B) none lengthdifferences. 5 bases) K1 UPf 1-6 (seq A) dye #1 UPr 1-6 (seq B) noneSeparate reactions have relative K2 UPf 1-6 (seq A + dye #1 UPr 1-6 (seqB) none mobility shifts from removal of cleavage site) nucleotides bycleavage within the primer. L1 UPf 1-6 (seq A) dye #1 UPr 1-6 (seq B)none Seperate reactions have relative L2 UPf 1-6 (seq A) dye #1 UPr 1-6(seq B) none mobility shifts resulting from L3 UPf 1-6 (seq A) dye #1UPr 1-6 (seq B) none staggered sample loading on the separation platformM1 UPf 1-6 (seq A) dye #1 UPr 1-6 (seq B) none Three seperate reactionsare pooled for M2 UPf 1-6 (seq A) dye #2 UPr 1-6 (seq B) none analysis.Resolution by size & M3 UPf 1-6 (seq A) dye #3 UPr 1-6 (seq B) nonefluorescence (multiplexing with dyes). (Note: products masses of thethree reactions overlap.)

[0277] TABLE 5 PRIMER SEQUENCES SEQ ID No. Accession # Primer PrimerName Primer Sequence SEQ ID No 1 X00351 beta-actin forward Sp6.1F1AGGTGACACTATAGAATAACCGA TAAGGCCAACCGCGAGAAGATGA SEQ ID No. 2 X00351beta-actin reverse T77R3 GTACGACTCACTATAGGGATGGA TAGCAACGTACATGGCTG SEQID No. 3 X00351 beta-actin reverse T77R3Pi GTACGACTCACTATAGGGATGGAPhosphorylated TAGCAACGTACATGGCTGPi SEQ ID No. 4 U02426 7.5 kb forwardSp6 (P2) F2 AGGTGACACTATAGAATAACTAT fragment GCCGGTATCAGCACC SEQ ID No.5 U02426 7.5 kb reverse T7 (P7) R2 GTACGACTCACTATAGGGAGATG fragmentGCAGCGTGATTTCAC SEQ ID No. 6 n/a INA D forward Sp6F1 (P2)AGGTGACACTATAGAATAGTGAC ACGTCGCAGAATGAG SEQ ID No. 7 n/a INA D reverseT7R1 (P7) GTACGACTCACTATAGGGATTGA CCCTTCAGTTGCTTGA SEQ ID No. 8 n/a hSPEforward Sp6F2 (P2) AGGTGACACTATAGAATAGCTTC ATTAGGTGGCTCAACA SEQ ID No. 9n/a hSPE reverse T7R2 (P7) GTACGACTCACTATAGGGAGGCT CAGCTTGTCGTAGTTC SEQID No. 10 n/a Survivin forward Sp6F1 (& F2) AGGTGACACTATAGAATAGTCAG (P2)CCCAACCTTCACATC SEQ ID No. 11 n/a Survivin reverse T7R2 (P7)GTACGACTCACTATAGGGACCAC CCTGCAGCTCTATGAC SEQ ID No. 12 n/a HNF 3 alphaSp6F3 (P2) AGGTGACACTATAGAATAACTTC forward AAGGCATACGAACAG SEQ ID No. 13n/a HNF 3 alpha T7R3 (P7) GTACGACTCACTATAGGGAGGGA reverseGCTAGGAAGTGTTTAG SEQ ID No. 14 M33197 GAPDH forward Sp6F1 (P2)AGGTGACACTATAGAATAAAGGT GAAGGTCGGAGTCAA SEQ ID No. 15 M33197 GAPDHreverse T7R1 (P7) GTACGACTCACTATAGGGAATGA CAAGCTTCCCGTTCTC SEQ ID No. 16M33197 GAPDH reverse T7RIPi (P7) GTACGACTCACTATAGGGAATGA phosphorylatedCAAGCTTCCCGTTCTCPi SEQ ID No. 17 n/a EST forward Sp6F4 (P2)AGGTGACACTATAGAATAGCTCA TCTGCCAACAATC SEQ ID No. 18 n/a EST reverse T7R4(P7) GTACGACTCACTATAGGGACTAG CGGAAGCAAATTACAC SEQ ID No. 19 n/a Hoxb 13forward Sp6F1 (P2) AGGTGACACTATAGAATAGCGAC ATGACTCCCTGTT SEQ ID No. 20n/a Hoxb 13 reverse T7R1 (& R2) GTACGACTCACTATAGGGAAACT (P7)TGTTAGCCGCATACTC SEQ ID No. 21 J01839 KanR forward Sp6 (P2)AGGTGACACTATAGAATAATCAT (V00359) (LP70) F2 CAGCATTGCATTCGATTCCTGTT TGSEQ ID No. 22 J01839 KanR reverse T7 (P7) R2 TACGACTCACTATAGGGAATTCC(V00359) GACTCGTCCAACATC SEQ ID No. 23 n/a Sp6 universalAGGTGACACTATAGAATA primer SEQ ID No. 24 n/a T7 universalGTACGACTCACTATAGGGA primer

[0278] The cases described above are provided for illustrative purposes.One skilled in the art can envision other embodiments that would achievethe general purpose of increasing sample throughput during separationand data collection.

[0279] While the foregoing invention has been described in some detailfor purposes of clarity and understanding, it will be clear to oneskilled in the art from a reading of this disclosure that variouschanges in form and detail can be made without departing from the truescope of the present invention. For example, all the techniques andcompositions described above may be used in various combinations. All ofthe compositions and/or methods disclosed and claimed herein can be madeand executed without undue experimentation in light of the presentdisclosure. While the compositions and methods of this invention havebeen described in terms of preferred embodiments, it will be apparent tothose of skill in the art that variations may be applied to thecompositions and/or methods, and in the steps or in the sequence ofsteps of the method described herein without departing from the concept,spirit and scope of the invention. More specifically, it will beapparent that certain agents which are both chemically andphysiologically related may be substituted for the agents describedherein while the same or similar results would be achieved. All suchsimilar substitutes and modifications apparent to those skilled in theart are deemed to be within the spirit, scope and concept of theinvention as defined by the appended claims. All publications, patents,patent applications, and/or other documents cited in this applicationare incorporated by reference in their entirety for all purposes to thesame extent as if each individual publication, patent, patentapplication, and/or other document were individually indicated to beincorporated by reference for all purposes.

1 30 1 46 DNA Artificial Sequence Description of Artificial SequencePrimer 1 aggtgacact atagaataac cgataaggcc aaccgcgaga agatga 46 2 41 DNAArtificial Sequence Description of Artificial Sequence Primer 2gtacgactca ctatagggat ggatagcaac gtacatggct g 41 3 41 DNA ArtificialSequence Description of Artificial Sequence Primer 3 gtacgactcactatagggat ggatagcaac gtacatggct g 41 4 38 DNA Artificial SequenceDescription of Artificial Sequence Primer 4 aggtgacact atagaataactatgccggta tcagcacc 38 5 38 DNA Artificial Sequence Description ofArtificial Sequence Primer 5 gtacgactca ctatagggag atggcagcgt gatttcac38 6 38 DNA Artificial Sequence Description of Artificial SequencePrimer 6 aggtgacact atagaatagt gacacgtcgc agaatgag 38 7 39 DNAArtificial Sequence Description of Artificial Sequence Primer 7gtacgactca ctatagggat tgacccttca gttgcttga 39 8 39 DNA ArtificialSequence Description of Artificial Sequence Primer 8 aggtgacactatagaatagc ttcattaggt ggctcaaca 39 9 39 DNA Artificial SequenceDescription of Artificial Sequence Primer 9 gtacgactca ctatagggaggctcagcttg tcgtagttc 39 10 38 DNA Artificial Sequence Description ofArtificial Sequence Primer 10 aggtgacact atagaatagt cagcccaacc ttcacatc38 11 39 DNA Artificial Sequence Description of Artificial SequencePrimer 11 gtacgactca ctatagggac caccctgcag ctctatgac 39 12 38 DNAArtificial Sequence Description of Artificial Sequence Primer 12aggtgacact atagaataac ttcaaggcat acgaacag 38 13 39 DNA ArtificialSequence Description of Artificial Sequence Primer 13 gtacgactcactatagggag ggagctagga agtgtttag 39 14 38 DNA Artificial SequenceDescription of Artificial Sequence Primer 14 aggtgacact atagaataaaggtgaaggtc ggagtcaa 38 15 39 DNA Artificial Sequence Description ofArtificial Sequence Primer 15 gtacgactca ctatagggaa tgacaagctt cccgttctc39 16 39 DNA Artificial Sequence Description of Artificial SequencePrimer 16 gtacgactca ctatagggaa tgacaagctt cccgttctc 39 17 36 DNAArtificial Sequence Description of Artificial Sequence Primer 17aggtgacact atagaatagc tcatctgcca acaatc 36 18 39 DNA Artificial SequenceDescription of Artificial Sequence Primer 18 gtacgactca ctatagggactagcggaagc aaattacac 39 19 36 DNA Artificial Sequence Description ofArtificial Sequence Primer 19 aggtgacact atagaatagc gacatgactc cctgtt 3620 39 DNA Artificial Sequence Description of Artificial Sequence Primer20 gtacgactca ctatagggaa acttgttagc cgcatactc 39 21 48 DNA ArtificialSequence Description of Artificial Sequence Primer 21 aggtgacactatagaataat catcagcatt gcattcgatt cctgtttg 48 22 38 DNA ArtificialSequence Description of Artificial Sequence Primer 22 tacgactcactatagggaat tccgactcgt ccaacatc 38 23 18 DNA Artificial SequenceDescription of Artificial Sequence Primer 23 aggtgacact atagaata 18 2419 DNA Artificial Sequence Description of Artificial Sequence Primer 24gtacgactca ctataggga 19 25 25 DNA Artificial Sequence Description ofArtificial Sequence Primer 25 tttttttagg tgacactata gaata 25 26 26 DNAArtificial Sequence Description of Artificial Sequence Primer 26tttttttgta cgactcacta taggga 26 27 21 DNA Artificial SequenceDescription of Artificial Sequence Primer 27 tagaggtgac actatagaat a 2128 21 DNA Artificial Sequence Description of Artificial Sequence Primer28 tttaggtgac actatagaat a 21 29 24 DNA Artificial Sequence Descriptionof Artificial Sequence Primer 29 gattagaggt gacactatag aata 24 30 18 DNAArtificial Sequence Description of Artificial Sequence Primer 30aggtgacact atagaata 18

What is claimed is:
 1. A method for analyzing gene expressioncomprising: a) obtaining a plurality of target sequences, wherein theplurality of target sequences comprises cDNA; b) multiplex amplifyingsaid plurality of target sequences, wherein multiplex amplifyingcomprises combining the plurality of target sequences, a plurality oftarget-specific primers, and one or more universal primers, therebyproducing a plurality of amplification products; c) separating one ormore members of the plurality of amplification products; d) detectingone or more members of the plurality of amplification products, therebygenerating a set of gene expression data; e) storing the set of geneexpression data in a database; and f) performing a comparative analysison the set of gene expression data, thereby analyzing the geneexpression.
 2. The method of claim 1, wherein obtaining the targetsequences comprises performing reverse transcription of mRNA.
 3. Themethod of claim 2, wherein the mRNA comprises mRNA derived from culturedcells.
 4. The method of claim 2, wherein said mRNA comprises mRNAderived from cultured cells subjected to a specific treatment.
 5. Themethod of claim 4, wherein said specific treatment comprises a chemicalexposure, an environmental stress, or an exposure to one or more viableorganisms or cells.
 6. The method of claim 1, wherein multiplexamplifying comprises simultaneously amplifying a plurality of cDNA inthe same reaction mixture; wherein said plurality of target-specificprimers comprises one or more target-specific primer pairs, each paircomprising a forward target-specific primer and a reversetarget-specific primer; and wherein the one or more universal-primerscomprises one or more universal primer pairs, each pair comprising aforward universal primer and a reverse universal primer.
 7. The methodof claim 1, wherein said plurality of target sequences further comprisesone or more reference sequences, wherein a portion of the one or morereference sequences is homologous to at least one member of theplurality of target-specific primers.
 8. The method of claim 7, whereinone or more of the reference sequences comprises sequences endogenouslypresent in the cDNA.
 9. The method of claim 7, wherein one or more ofthe reference sequences comprises sequences exogenously added to thecDNA.
 10. The method of claim 1, wherein at least one member of theplurality of target-specific primers or universal primers furthercomprises a modified nucleotide.
 11. The method of claim 10, wherein themodified nucleotide prevents amplification of one or more portions ofthe at least one member of the plurality of target-specific primers oruniversal primers
 12. The method of claim 10, wherein the modifiednucleotide comprises one or more non-nucleotide linkers, alkyl chains,or abasic nucleotides.
 13. The method of claim 1, wherein at least onemember of the plurality of target-specific primers or universal primersfurther comprises a cleavable linker.
 14. The method of claim 1, whereinat least one universal primer further comprises a label.
 15. The methodof claim 14, wherein the label comprises one or more of a chromaphore, afluorophore, a dye, a releasable label, a mass label, an affinity label,a friction moiety, a hydrophobic group, or an isotopic label.
 16. Themethod of claim 1, wherein each member of the plurality oftarget-specific primers comprises a first sequence that is derived froma target gene of interest and positioned within a 3′ region of themember, and a second sequence that is complementary to the universalprimer and positioned within a 5′ region of the member.
 17. The methodof claim 1, wherein the one or more universal primers comprise one ormore semi-universal primers.
 18. The method of claim 17, wherein the oneor more semi-universal primers comprise primers which are complementaryto one or more forward target-specific primers, one or more reversetarget-specific primers, or a combination thereof.
 19. The method ofclaim 18, wherein the one or more semi-universal primers comprise afirst semi-universal primer that is complementary to all of the one ormore forward target-specific primers, and a second semi-universal primerthat is complementary to all of the one or more reverse target-specificprimers.
 20. The method of claim 17, wherein each of the one or moresemi-universal primers comprises a unique label.
 21. The method of claim1, wherein multiplex amplifying comprises providing the universal primerin an excess concentration relative to the target-specific primer. 22.The method of claim 21, wherein a universal primer: target-specificprimer concentration ratio ranges from about 5: 1 to about 100:1. 23.The method of claim 21, wherein a universal primer: target-specificprimer concentration ratio is about 10:1.
 24. The method of claim 1,wherein an annealing temperature of the universal primer is higher thanan annealing temperature of the target-specific primer.
 25. The methodof claim 1, wherein obtaining a plurality of target sequences comprisesproviding two or more target sequences having two or moretarget-specific primer annealing temperatures.
 26. The method of claim1, wherein multiplex amplifying the cDNA comprises amplifying targetgenes that have comparable expression levels.
 27. The method of claim 1,wherein multiplex amplifying the cDNA comprises attenuating anamplification of abundant target genes.
 28. The method of claim 27,wherein attenuating the amplification of abundant target genes comprisesusing one or more modified target-specific primers.
 29. The method ofclaim 28, wherein the one or more modified target-specific primercomprises a blocking group attached at a 3′ end of the modifiedtarget-specific primer.
 30. The method of claim 28, wherein the one ormore modified target-specific primer comprises one or more abasicnucleotides or mismatch nucleotides.
 31. The method of claim 28, whereinusing one or more modified target-specific primers comprises providing amixture of the one or more modified target-specific primers with one ormore unmodified target-specific primers, at a ratio optimized for adesired amount of attenuation.
 32. The method of claim 28, wherein theone or more modified target-specific primer comprises a blocking groupattached at a 3′ end of a reverse target-specific primer.
 33. The methodof claim 28, wherein the one or more modified target-specific primerscomprise primers having a phosphate group on the terminal 3′-hydroxyl ofthe target-specific primer.
 34. The method of claim 28, wherein the oneor more modified target-specific primers comprise primers having anucleotide penultimate to the terminal 3′-nucleotide and attached via a3′-3′ phosphodiester linkage.
 35. The method of claim 1, whereinmultiplex amplifying further-comprises altering the length of one ormore of the universal primers or one or more of the plurality oftarget-specific primers prior to combining.
 36. The method of claim 35,wherein altering the length comprises adding nucleotides to an end of auniversal primer or a target-specific primer.
 37. The method of claim35, wherein altering the length comprises inserting nucleotides within auniversal primer or a target-specific primer.
 38. The method of claim37, wherein altering the length of a target-specific primer comprisesinserting nucleotides between a universal sequence and a target-specificsequence of the target-specific primer.
 39. The method of claim 35,wherein altering the length comprises incorporating a non-nucleotidelinker into a universal primer or a target-specific primer.
 40. Themethod of claim 35, wherein altering the length comprises cleaving theone or more universal primers or the one or more target-specificprimers.
 41. The method of claim 35, wherein one or more of theuniversal primers or one or more of the plurality of target-specificprimers comprise semi-universal primers.
 42. The method of claim 1,wherein the plurality of amplification products comprises a plurality oflabels at predetermined molar ratios.
 43. The method of claim 42,wherein the plurality of labels is incorporated on a singleoligonucleotide primer.
 44. The method of claim 42, wherein theplurality of labels is incorporated on a plurality of oligonucleotideprimers.
 45. The method of claim 1, wherein separating the one or moremembers of the plurality of amplification products comprises performingone or more size separation techniques.
 46. The method of claim 45,wherein separating the one or more members of the plurality ofamplification products comprises performing mass spectrometry.
 47. Themethod of claim 45, wherein separating the one or more members of theplurality of amplification products comprises employing anelectrophoresis platform.
 48. The method of claim 47, wherein theelectrophoresis platform comprises one or more of a capillary platform,a microcapillary platform, a microfluidics platform, an agarose gel, anacrylamide gel, an agarose/acrylamide gel or a chromatographic platform.49. The method of claim 45, wherein separating the one or more membersof the plurality of amplification products comprises performing HPLC orFPLC.
 50. The method of claim 1, wherein separating the one or moremembers of the plurality of amplification products comprises performingHPLC followed by mass spectroscopy.
 51. The method of claim 1, whereindetecting the one or more members of the plurality of amplificationproducts comprises measuring one or more inherent properties of theamplification products.
 52. The method of claim 51, wherein the one ormore inherent properties comprise mass, light absorption, or anelectrochemical property.
 53. The method of claim 1, wherein detectingthe one or more members of the plurality of amplification productscomprises measuring the presence, absence, or quantity of a labeledamplification product.
 54. The method of claim 53, wherein the labeledamplification product comprises a singly labeled amplification product,a multiply-labeled amplification product, or a combination thereof. 55.The method of claim 53, wherein detecting comprises resolving a firstsignal from a singly labeled amplification product and a second signalfrom a multiply labeled amplification product by deconvolution of thedata.
 56. The method of claim 53, wherein detecting comprises resolvinga first signal from a singly labeled amplification product and a secondsignal from a multiply labeled amplification product by reciprocalsubtraction of the first or second signal from an overlapping signal.57. The method of claim 1, wherein performing the comparative analysiscomprises measuring a ratio of each target gene to each reference gene.58. The method of claim 1, wherein one or more of the multiplexamplifying, separating and detecting is performed in a high throughputformat.
 59. A method for analyzing gene expression comprising: a)obtaining cDNA from a plurality of samples for a plurality of targetsequences; b) performing a plurality of multiplexed amplifications ofthe target sequences, thereby producing a plurality of multiplexedamplification products; c) pooling the plurality of multiplexedamplification products; d) separating the plurality of multiplexedamplification products; e) detecting the plurality of multiplexedamplification products, thereby generating a set of gene expressiondata; f) storing the set of gene expression data in a database; and g)performing a comparative analysis of the set of gene expression data.60. The method of claim 59, wherein performing the plurality ofmultiplexed amplifications comprises combining the plurality of targetsequences, one or more target-specific primers, and one or moreuniversal primers
 61. The method of claim 60, wherein at least one ofthe one or more universal primers or one or more target-specific primerscomprises a label.
 62. The method of claim 61, wherein a firstmultiplexed amplification is performed with a primer comprising a firstlabel that produces a first signal, and a second multiplexedamplification is performed with a primer comprising a second label thatproduces a second signal, wherein the first and second signals aredistinguishable from one another.
 63. The method of claim 62, whereinthe first and second signals are distinguishable by deconvolution ofsignals obtained from the plurality of multiplexed amplificationproducts.
 64. The method of claim 61, wherein the first or second labelcomprises a high-affinity intercalating dye.
 65. The method of claim 60,wherein performing the plurality of amplifications of the targetsequences comprises using universal primers having two or more lengths,and wherein detecting the plurality of multiplexed amplificationproducts comprises measuring one or more size shifts among the pluralityof multiplexed amplification products.
 66. The method of claim 60,wherein performing the plurality of amplifications of the targetsequences comprises using target-specific primers having two or morelengths, and wherein detecting the plurality of multiplexedamplification products comprises measuring one or more size shifts amongthe plurality of multiplexed amplification products.
 67. The method ofclaim 66, performing the plurality of amplifications of the targetsequences comprises using universal primers comprising one or morecleavage sites, and wherein detecting the plurality of multiplexedamplification products comprises measuring one or more size shifts amongthe plurality of multiplexed amplification products.
 68. The method ofclaim 60, wherein separating the plurality of multiplexed amplificationproducts comprises shifting the mobility of member amplificationproducts relative to one another.
 69. The method of claim 68, whereinshifting the mobility comprises incorporating a friction moiety into oneor more of the universal primers, thereby creating a reduction inmobility of the amplification products.
 70. The method of claim 59,wherein separating comprises applying each set of multiplexamplification products to a separation platform at different times. 71.The method of claim 59, wherein performing the plurality ofamplifications comprises performing a polymerase chain reaction, atranscription-based amplification, a self-sustained sequencereplication, a nucleic acid sequence based amplification, a ligase chainreaction, a ligase detection reaction, a strand displacementamplification, a repair chain reaction, a cyclic probe reaction, a rapidamplification of cDNA ends, an invader assay, a solid phase assay, asolution phase assay, or a combination thereof.
 72. The method of claim71, wherein the solid phase assay comprises a bridge amplification orrolling circle amplification.
 73. The method of claim 59, wherein one ormore of the performing, separating and detecting is performed in a highthroughput format.
 74. The method of claim 73, wherein one or more ofthe performing, separating and detecting steps is performed at a rate ofabout 1000 samples per hour.
 75. A method for analyzing gene expressioncomprising: a) obtaining cDNA from multiple samples; b) amplifying aplurality of target sequences from the cDNA, thereby producing amultiplex set of amplification products; c) separating and detecting theamplification products using a high throughput platform, whereindetecting generates a set of gene expression data; and d) storing theset of gene expression data in a database; and e) performing acomparative analysis of the set of gene expression data.
 76. The methodof claim 75, wherein amplifying the plurality of target sequencescomprises using one or more universal primers.
 77. The method of claim75, wherein amplifying the plurality of target sequences comprises usingone or more target-specific primers.
 78. The method of claim 77, whereinthe one or more universal primers or the one or more target-specificprimers comprise one or more non-nucleotide linkers.
 79. The method ofclaim 75, wherein separating and detecting the amplification productscomprises performing mass spectrometry, polyacrylamide gelelectrophoresis, HPLC, capillary electrophoresis, microcapillaryelectrophoresis, or a combination thereof.
 80. The method of claim 75,wherein separating and detecting the amplification products is performedusing microfluidic devices.
 81. The method of claim 75, wherein the highthroughput platform comprises an HPLC for separating the amplificationproducts and a mass spectrometer for detecting the amplificationproducts.
 82. The method of claim 75, wherein the high throughputplatform comprises one or more miniaturized scale platforms.
 83. Themethod of claim 75, wherein one or more of the amplifying, separatingand detecting steps is performed at a rate of about 100 samples per hourto about 5,000 samples per hour.
 84. The method of claim 75, wherein oneor more of the amplifying, separating and detecting steps is performedat a rate of about 1000 samples per hour.
 85. The method of claim 75,wherein amplifying the plurality of target sequences comprisesperforming on or more of a polymerase chain reaction, atranscription-based amplification, a self-sustained sequencereplication, a nucleic acid sequence based amplification, a ligase chainreaction, a ligase detection reaction, a strand displacementamplification, a repair chain reaction, a cyclic probe reaction, a rapidamplification of cDNA ends, an invader assay, a solution phaseamplification assay, or a solid phase amplification assay.
 86. Themethod of claim 85, wherein the solid phase amplification assaycomprises a bridge amplification or rolling circle amplification.
 87. Apool of amplification products prepared by the method of claim
 1. 88. Apool of amplification products prepared by the method of claim
 59. 89. Apool of amplification products prepared by the method of claim
 75. 90. Asystem for analyzing gene expression, the system comprising: a) anamplification module for producing a plurality of amplification productsfrom a pool of target sequences, the amplification module comprising atleast one pair of universal primers and at least one pair oftarget-specific primers; b) a detection module for detecting one or moremembers of the plurality of amplification products, wherein thedetection module detects a presence, absence, or quantity of the one ormore members, and generates a set of gene expression data comprising aplurality of data points; and c) an analyzing module in operationalcommunication with the detection module, the analyzing module comprisinga computer or computer-readable medium comprising one or more logicalinstructions which organize the plurality of data points into a databaseand one or more logical instructions which analyze the plurality of datapoints.
 91. The system of claim 90, wherein one or more of theamplification module, the detection module, and the analyzing module acomprise high throughput system.
 92. The system of claim 90, wherein theat least one pair of universal primers or the at least one pair oftarget-specific primers comprise one or more abasic nucleotides.
 93. Thesystem of claim 90, wherein the amplification module comprises a uniquepair of universal primers for each target sequence.
 94. The system ofclaim 90, wherein the amplification module comprises components toperform a polymerase chain reaction, a transcription-basedamplification, a self-sustained sequence replication, a nucleic acidsequence based amplification, a ligase chain reaction, a ligasedetection reaction, a strand displacement amplification, a repair chainreaction, a cyclic probe reaction, a rapid amplification of cDNA ends,an invader assay, a solid phase amplification reaction, a solution phaseamplification reaction, or a combination thereof.
 95. The system ofclaim 90, wherein the detection module comprises a mass spectrometer.96. The system of claim 90, wherein the detection module comprises anelectrophoretic device.
 97. The system of claim 90, wherein the one ormore logical instructions for analyzing the plurality of data pointscomprises software for generating a graphical representation of theplurality of data points.
 98. The system of claim 90, wherein the one ormore logical instructions which analyze the plurality of data points areembodied in system software which performs combinatorial analysis on theplurality of data points.
 99. The system of claim 90, wherein the one ormore logical instructions for analyzing the plurality of data pointscomprises software for performing difference analysis upon the pluralityof data points.
 100. The system of claim 90, the analyzing modulefurther comprising an output file.
 101. A composition for preparing aplurality of amplification products from a plurality of mRNA targetsequences, the composition comprising: one or more pairs of universalprimers; and one or more pairs of target-specific primers, wherein thetarget-specific primers comprise one or more regions complementary tothe one or more pairs of universal primers and one or more regionscomplementary to one or more target mRNA sequences.
 102. The compositionof claim 101, wherein one or more members of the one or more pairs ofuniversal primers or one or more pairs of target-specific primerscomprises a non-nucleotide linkage.
 103. The composition of claim 101,wherein one or more members of the one or more pairs of universalprimers or one or more pairs of target-specific primers comprise one ormore cleavable nucleotides.
 104. A kit for obtaining a multiplex set ofamplification products of target genes and references-genes, the kitcomprising: a) at least one pair of universal primers; b) at least onepair of target-specific primers; c) at least one pair of referencegene-specific primers; and d) one or more amplification reactionenzymes, reagents, or buffers.
 105. The kit of claim 83, furthercomprising: e) software for storing and analyzing data obtained from theamplification reactions.
 106. The kit of claim 83, wherein theuniversal-primers comprises labeled primers.
 107. The kit of claim 83,wherein the simultaneous amplification of the reference-genes allowsquantitation of the amplification products.